Cell-specific Signatures Research Articles

Modelling T cell proliferation: Dynamics heterogeneity depending on cell differentiation, age, and genetic background.

Cell proliferation is the common characteristic of all biological systems. The immune system insures the maintenance of body integrity on the basis of a continuous production of diversified T lymphocytes in the thymus. This involves processes of proliferation, differentiation, selection, death and migration of lymphocytes to peripheral tissues, where proliferation also occurs upon antigen recognition. Quantification of cell proliferation dynamics requires specific experimental methods and mathematical modelling. Here, we assess the impact of genetics and aging on the immune system by investigating the dynamics of proliferation of T lymphocytes across their differentiation through thymus and spleen in mice. Our investigation is based on single-cell multicolour flow cytometry analysis revealing the active incorporation of a thymidine analogue during S phase after pulse-chase-pulse experiments in vivo, versus cell DNA content. A generic mathematical model of state transition simulates through Ordinary Differential Equations (ODEs) the evolution of single cell behaviour during various durations of labelling. It allows us to fit our data, to deduce proliferation rates and estimate cell cycle durations in sub-populations. Our model is simple and flexible and is validated with other durations of pulse/chase experiments. Our results reveal that T cell proliferation is highly heterogeneous but with a specific “signature” that depends upon genetic origins, is specific to cell differentiation stages in thymus and spleen and is altered with age. In conclusion, our model allows us to infer proliferation rates and cell cycle phase durations from complex experimental 5-ethynyl-2'-deoxyuridine (EdU) data, revealing T cell proliferation heterogeneity and specific signatures.

Two-Photon Intravital Fluorescence Lifetime Imaging of the Kidney Reveals Cell-Type Specific Metabolic Signatures.

In the live animal, tissue autofluorescence arises from a number of biologically important metabolites, such as the reduced form of nicotinamide adenine dinucleotide. Because autofluorescence changes with metabolic state, it can be harnessed as a label-free imaging tool with which to study metabolism in vivo Here, we used the combination of intravital two-photon microscopy and frequency-domain fluorescence lifetime imaging microscopy (FLIM) to map cell-specific metabolic signatures in the kidneys of live animals. The FLIM images are analyzed using the phasor approach, which requires no prior knowledge of metabolite species and can provide unbiased metabolic fingerprints for each pixel of the lifetime image. Intravital FLIM revealed the metabolic signatures of S1 and S2 proximal tubules to be distinct and resolvable at the subcellular level. Notably, S1 and distal tubules exhibited similar metabolic profiles despite apparent differences in morphology and autofluorescence emission with traditional two-photon microscopy. Time-lapse imaging revealed dynamic changes in the metabolic profiles of the interstitium, urinary lumen, and glomerulus-areas that are not resolved by traditional intensity-based two-photon microscopy. Finally, using a model of endotoxemia, we present examples of the way in which intravital FLIM can be applied to study kidney diseases and metabolism. In conclusion, intravital FLIM of intrinsic metabolites is a bias-free approach with which to characterize and monitor metabolism in vivo, and offers the unique opportunity to uncover dynamic metabolic changes in living animals with subcellular resolution.

Abstract P5-05-01: Physico-biochemical regulation of EMT by microtubule associated protein 7 (MAP7)

Abstract Background: We previously reported about 500 cancer stem cell (CSC) specific gene signatures from patient tumor samples. After screening with shRNAs for the 500 genes affecting mammosphere forming ability, we identified microtubule-assoicated protein 7 (MAP7) as one of the top candidate genes, which may serve as a target for breast CSCs. Although MAP7 is a predominant epithelial microtubule binding protein, only limited number of reports suggests that MAP7 may be a regulator of microtubule dynamics during cell division and a cofactor of Kinesin-1 in compartment transport in cells. However, little is known about how MAP7 supports epithelial cancers, especially breast cancer. Previously, we have reported that the mammosphere forming cells exhibit treatment resistance and high metastatic potential, which are intrinsic characters for CSCs displaying epithelial mesenchymal transition (EMT). We hypothesize that MAP7 supports breast cancer progression by promoting CSC self-renewal and survival through regulation of EMT. Objectives: Here, we aim to show that MAP7 is an essential regulator of breast CSCs and to elucidate mechanism behind EMT regulation by MAP7 in breast CSCs. Methods and Results: On Oncomine database analysis, MAP7 was up-regulated in most epithelial cancers, when compared to the corresponding normal tissues. Similarly, its expression in breast cancer was 2-fold higher than in the normal breast tissue (p<0.05), but without significant variances in the expression across the breast cancer subtypes. Gene silencing of MAP7 significantly reduced CD44+/CD24- breast CSC populations and mammosphere forming efficiencies of MDA-MB-231, HCC1937, and MDA-MB-468 breast cancer cells. Furthermore, the silencing of MAP7 expression compromised invasive potential of MDA-MB-231 cells by 50% and significantly altered the cell membrane mechanics of MDA-MB-468 cells, as indicated by a high-content image analysis for cell shapes and cell adhesion efficiency. More importantly, delivery of siRNA in vivo inhibited the growth of BCM2147 patient-derived tumor, and limiting dilution assay demonstrated that the tumor initiation potential of BCM2147 can be eliminated by MAP7 silencing. Through confocal microscope analysis of images of fluorescent immunostaining and co-immunoprecipatation assays, MAP7 showed polarized-expressions in spindle-shaped cancer cells and was co-localized with Focal Adhesion Kinase (FAK). Moreover, MAP7 silencing inhibited the phosphorylation of FAK by inactivating p130CAS and JSAP1, the upper stream and the down-stream regulators of FAK. Conclusion: We have showed the ectopic expression of MAP7 in breast tumors and other epithelial tumors, suggesting MAP7 may be involved in tumorigenesis and critical for the survival of tumor cells. Moreover, our results suggest that MAP7 is a key element for survival and self-renewal of breast CSCs through polarization of cells and activation of FAK, required for the initiation of EMT. To that end, here we report that MAP7 is essential for breast cancer growth by supporting CSC survival and self-renewal. Citation Format: Choi DS, Dave B, Rosato RR, Chang JC. Physico-biochemical regulation of EMT by microtubule associated protein 7 (MAP7) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-05-01.

Dynamic Alterations in Gene Expression in Ibrutinib Treated CLL Reveal Profound Impact on Multiple Signaling Pathways

Ibrutinib covalently binds Bruton tyrosine kinase (BTK) leading to sustained target inhibition. On-target effects include reductions in BCR and NF-κB signaling and inhibition of tumor proliferation and survival (Herman, Blood 2014). In randomized trials, ibrutinib induced superior responses and extended the survival of patients with chronic lymphocytic leukemia (CLL) in both the relapsed/refractory and frontline setting (Byrd, NEJM 2014; Burger, NEJM 2015). However, deep remissions are uncommon and most patients have readily detectable residual disease in the blood. To better understand the biology of persistent disease on ibrutinib, we performed RNA-sequencing on CD19+ selected tumor cells from 14 of 86 patients treated with single-agent ibrutinib (ClinicalTrials.gov NCT01500733); 8/14 (57%) had an unmutated IGVHsequence, 8/14 (57%) had del(17p), 6/14 (43%) were previously untreated and 4/14 (29%) had relapsed on ibrutinib at the time of analysis (range: 8-47 months). At best response, 3/14 (21%) achieved a CR, 10/14 (71%) a PR or PRL and 1/14 (7%) had SD. Three samples per patient were analyzed: T1 (baseline), T2 (day 28) and T3 (6 months), for a total of 42 samples. Principal component analysis (PCA) grouped samples by patient, as observed in a similar study (Herishanu, Blood 2011). After adjusting for patient-specific effects, PCA clearly revealed the effect of treatment, separating on-treatment samples (T2, T3) from baseline (T1). To identify differentially expressed (DE) genes, we selected transcripts that showed at least a 2-fold change between baseline and treatment samples (FDR<0.1). Of the ~14,000 measured genes, there were 653 DE genes on-treatment compared to baseline. Notably, T3 samples demonstrated stronger changes in gene expression than T2 samples, in line with a more profound treatment response with continued drug exposure. Compared to T1, there were 80 DE genes expressed at T2 only, 330 at T3 only, and 243 at both T2 and T3. Among all DE genes, 498 decreased and 155 increased at either T2 or T3 compared to T1.To identify the biologic impact of treatment, we dissected the gene expression changes using pathway-specific gene signatures represented in the KEGG pathways with the molecular signatures database tool (MSigDB; Broad Institute, Boston, MA). Strikingly, no pathways were significantly enriched on treatment. In contrast, multiple gene signatures (n=46) were significantly turned down on ibrutinib (FDR<0.01). Using more stringent cutoffs (p<0.0005; FDR q<0.005; ≥5 significant genes per pathway), nine KEGG pathways were significantly inhibited by ibrutinib, including cytokine signaling, cell adhesion, systemic lupus, p53 response, MAPK signaling, cell cycle, focal adhesion, calcium signaling, and Wnt signaling. We also applied the same analysis to experimentally-derived B cell-specific gene signatures (Shaffer, Immunity 2001; Herishanu, Blood 2011), demonstrating significant inhibition of both BCR and NF-κB signaling pathways. We derived signature scores by averaging the expression of the signature-specific genes and estimated the degree of pathway inhibition from the ratio of signature averages in treated versus baseline samples. Of the 11 significantly altered pathways, all demonstrated >50% reduction in signature scores, ranging from a 71% reduction for BCR to 54% for p53. The degree of inhibition of the signaling pathways was independent of biologic and clinical features, including IGVH mutation status, presence or absence of del(17p), and prior treatment status.In summary, ibrutinib induces widespread changes in gene expression in CLL cells in vivo reflecting strong inhibitory effects across multiple pathways that suggest a broader impact on tumor biology than expected from pure loss of BTK function. The surprising extent of changes across multiple diverse pathways could reflect inhibition of tumor-microenvironment crosstalk and consequent changes in the tumor milieu. This notion is consistent with the more profound inhibition observed with longer treatment duration. Equally notable, we found no distinct biologic signature(s) upregulated in response to ibrutinib which may indicate the absence of a cellular stress response and is consistent with the apparent resting state of the residual tumor and the observed slow attrition of cells over time.This research was supported by the Intramural Research Program of the NHLBI. DisclosuresFarooqui:Merck: Employment. Wu:Neon Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder. Wiestner:Pharmacyclics: Research Funding; Acerta Pharma: Research Funding.

Silencing of GATA3 defines a novel stem cell-like subgroup of ETP-ALL.

BackgroundGATA3 is pivotal for the development of T lymphocytes. While its effects in later stages of T cell differentiation are well recognized, the role of GATA3 in the generation of early T cell precursors (ETP) has only recently been explored. As aberrant GATA3 mRNA expression has been linked to cancerogenesis, we investigated the role of GATA3 in early T cell precursor acute lymphoblastic leukemia (ETP-ALL).MethodsWe analyzed GATA3 mRNA expression by RT-PCR (n = 182) in adult patients with T-ALL. Of these, we identified 70 of 182 patients with ETP-ALL by immunophenotyping. DNA methylation was assessed genome wide (Illumina Infinium® HumanMethylation450 BeadChip platform) in 12 patients and GATA3-specifically by pyrosequencing in 70 patients with ETP-ALL. The mutational landscape of ETP-ALL with respect to GATA3 expression was investigated in 18 patients and validated by Sanger sequencing in 65 patients with ETP-ALL. Gene expression profiles (Affymetrix Human genome U133 Plus 2.0) of an independent cohort of adult T-ALL (n = 83) were used to identify ETP-ALL and investigate GATA3low and GATA3high expressing T-ALL patients. In addition, the ETP-ALL cell line PER-117 was investigated for cytotoxicity, apoptosis, GATA3 mRNA expression, DNA methylation, and global gene expression before and after treatment with decitabine.ResultsIn our cohort of 70 ETP-ALL patients, 33 % (23/70) lacked GATA3 expression and were thus defined as GATA3low. DNA methylation analysis revealed a high degree of GATA3 CpG island methylation in GATA3low compared with GATA3high ETP-ALL patients (mean 46 vs. 21 %, p < 0.0001). Genome-wide expression profiling of GATA3low ETP-ALL exhibited enrichment of myeloid/lymphoid progenitor (MLP) and granulocyte/monocyte progenitor (GMP) genes, while T cell-specific signatures were downregulated compared to GATA3high ETP-ALL. Among others, FLT3 expression was upregulated and mutational analyses demonstrated a high rate (79 %) of FLT3 mutations. Hypomethylating agents induced reversal of GATA3 silencing, and gene expression profiling revealed downregulation of hematopoietic stem cell genes and upregulation of T cell differentiation.ConclusionsWe propose GATA3low ETP-ALL as a novel stem cell-like leukemia with implications for the use of myeloid-derived therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0324-8) contains supplementary material, which is available to authorized users.

Proteomics of Human Dendritic Cell Subsets Reveals Subset-Specific Surface Markers and Differential Inflammasome Function

SummaryDendritic cells (DCs) play a key role in orchestrating adaptive immune responses. In human blood, three distinct subsets exist: plasmacytoid DCs (pDCs) and BDCA3+ and CD1c+ myeloid DCs. In addition, a DC-like CD16+ monocyte has been reported. Although RNA-expression profiles have been previously compared, protein expression data may provide a different picture. Here, we exploited label-free quantitative mass spectrometry to compare and identify differences in primary human DC subset proteins. Moreover, we integrated these proteomic data with existing mRNA data to derive robust cell-specific expression signatures with more than 400 differentially expressed proteins between subsets, forming a solid basis for investigation of subset-specific functions. We illustrated this by extracting subset identification markers and by demonstrating that pDCs lack caspase-1 and only express low levels of other inflammasome-related proteins. In accordance, pDCs were incapable of interleukin (IL)-1β secretion in response to ATP.

Immunogenic Subtypes of Breast Cancer Delineated by Gene Classifiers of Immune Responsiveness.

The abundance and functional orientation of tumor-infiltrating lymphocytes in breast cancer is associated with distant metastasis-free survival, yet how this association is influenced by tumor phenotypic heterogeneity is poorly understood. Here, a bioinformatics approach defined tumor biologic attributes that influence this association and delineated tumor subtypes that may differ in their ability to sustain durable antitumor immune responses. A large database of breast tumor expression profiles and associated clinical data was compiled, from which the ability of phenotypic markers to significantly influence the prognostic performance of a classification model that incorporates immune cell-specific gene signatures was ascertained. Markers of cell proliferation and intrinsic molecular subtype reproducibly distinguished two breast cancer subtypes that we refer to as immune benefit-enabled (IBE) and immune benefit-disabled (IBD). The IBE tumors, comprised mostly of highly proliferative tumors of the basal-like, HER2-enriched, and luminal B subtypes, could be stratified by the immune classifier into significantly different prognostic groups, while IBD tumors could not, indicating the potential for productive engagement of metastasis-protective immunity in IBE tumors, but not in IBD tumors. The prognostic stratification in IBE was independent of conventional variables. Gene network analysis predicted the activation of TNFα/IFNγ signaling pathways in IBE tumors and the activation of the transforming growth factor-β pathway in IBD tumors. This prediction supports a model in which breast tumors can be distinguished on the basis of their potential for metastasis-protective immune responsiveness. Whether IBE and IBD represent clinically relevant contexts for evaluating sensitivity to immunotherapeutic agents warrants further investigation. Cancer Immunol Res; 4(7); 600-10. ©2016 AACR.

Transcriptional profiling of mast cell and basophil responses to differential stimuli identifies unique gene signatures.

Abstract Mast cells (MC) and basophils (BΦ) are key effector cells in allergy that are developmentally related. Functionally, they both release mediators like histamine and granule proteases. However, studies also suggest that MC and BΦ have non-redundant roles in allergic diseases. Their ability to respond to IL-33—an epithelium-derived cytokine that helps initiate allergic inflammation—is not well understood. To fully characterize the transcriptional responses and heterogeneity of MC and BΦ, we performed large-scale comparative microarrays of bone marrow-derived mast cells (BMMCs) and basophils (BMBs) either at rest, upon IgE activation, or upon IL-33 activation. Hierarchical clustering demonstrated that BMMCs had activation-specific transcriptional signatures. IgE-crosslinking upregulated 1803 unique genes including Ccl7, Fxyd6, and Cd33; while IL-33 stimulation induced 1918 genes including Il1b, Cd72, and Tnfaip2. Focused bioinformatics pathway analysis demonstrated IgE-activation aligned with pathways relating to complement activation and CD137 signaling, while IL-33 initiated responses in the NF-κB and IL-10 signaling pathways. Furthermore, BMBs activated via IgE-crosslinking induced type 2 immune response genes like Il2, Il4, Ccl17 and Il13 which were surprisingly absent in IL-33 stimulated BMBs. Further analysis revealed cell-specific transcriptional signatures with 4360 genes that were significant in BMBs but not in BMMCs after IgE activation, and 1645 after IL-33 stimulation. Here we show that MC and BΦ have cell-specific and activation-specific transcriptional responses, and our data suggest novel gene networks and pathways that may shape how the immune system responds to allergens and innate cytokines.

DNA context represents transcription regulation of the gene in mouse embryonic stem cells.

Understanding gene regulatory information in DNA remains a significant challenge in biomedical research. This study presents a computational approach to infer gene regulatory programs from primary DNA sequences. Using DNA around transcription start sites as attributes, our model predicts gene regulation in the gene. We find that H3K27ac around TSS is an informative descriptor of the transcription program in mouse embryonic stem cells. We build a computational model inferring the cell-type-specific H3K27ac signatures in the DNA around TSS. A comparison of embryonic stem cell and liver cell-specific H3K27ac signatures in DNA shows that the H3K27ac signatures in DNA around TSS efficiently distinguish the cell-type specific H3K27ac peaks and the gene regulation. The arrangement of the H3K27ac signatures inferred from the DNA represents the transcription regulation of the gene in mESC. We show that the DNA around transcription start sites is associated with the gene regulatory program by specific interaction with H3K27ac.

Epigenetic Control of Smooth Muscle Cell Identity and Lineage Memory.

Vascular smooth muscle cells (SMCs), like all cells, acquire a cell-specific epigenetic signature during development that includes acquisition of a unique repertoire of histone and DNA modifications. These changes are postulated to induce an open chromatin state (referred to as euchromatin) on the repertoire of genes that are expressed in differentiated SMC, including SMC-selective marker genes like Acta2 and Myh11, as well as housekeeping genes expressed by most cell types. In contrast, genes that are silenced in differentiated SMC acquire modifications associated with a closed chromatin state (ie, heterochromatin) and transcriptional silencing. Herein, we review mechanisms that regulate epigenetic control of the differentiated state of SMC. In addition, we identify some of the major limitations in the field and future challenges, including development of innovative new tools and approaches, for performing single-cell epigenetic assays and locus-selective editing of the epigenome that will allow direct studies of the functional role of specific epigenetic controls during development, injury repair, and disease, including major cardiovascular diseases, such as atherosclerosis, hypertension, and microvascular disease, associated with diabetes mellitus.

295. Flow-Based Cell-Specific Interferon Signature as a Biomarker in Systemic Lupus Erythematosus

295. Flow-Based Cell-Specific Interferon Signature as a Biomarker in Systemic Lupus Erythematosus

Abstract 2359: Bioinformatics analysis of an Ad-VEGF flank angiogenesis model identifies vessel subtype gene signatures: Implications for anti-VEGF therapy

Abstract Vascular Endothelial Growth Factor A (VEGF-A) is the predominant growth factor expressed by tumor cells to drive angiogenesis and solid tumor growth. Antiangiogenesis therapies have been clinically demonstrated to be effective, however, there are still not effective means to preselect those patients most likely to derive optimal benefit. To identify blood vessel markers that may differentiate patients responsive to VEGF pathway blockade, we used a well-established in vivo model to generate pathological angiogenesis following the delivery of adenovirus engineered to express VEGF-A164 (Ad-VEGF) to a localized subcutaneous site within the flank of a nude mouse. This model provides a temporally controlled induction of angiogenesis and subsequent remodeling and maturation of the vasculature. This enabled us to study discrete subclasses of vessel which have been previously shown to have differential sensitivities to anti-angiogenic therapy. Here we report gene expression profiling on a time course of the Ad-VEGF flank model which was used to characterize molecular and genetic changes associated with these distinct vessel subtypes. Using a nearest centroid-based classification algorithm we identified unique endothelial cell specific gene signatures representing ‘early’, ‘middle' and ‘late' stages of vessel development and maturation. To evaluate the utility of these gene signatures in predicting sensitivity to anti-VEGF therapy, we profiled a publically available gene array dataset generated from a mouse xenograft model following anti-VEGF treatment with our vessel subtype gene signatures. A majority of the early and middle vessel genes were significantly reduced in bevacizumab-treated animals, demonstrating they respond to VEGF pathway inhibition. To further evaluate the potential value of these gene signatures as predictive biomarkers we profiled a dataset of 248 gastric tumors using hierarchical clustering. Our analysis shows the middle vessel signature was highly expressed in a mesenchymal molecular subtype of gastric cancer. Interestingly, the majority of the mesenchymal gastric tumors were ‘diffuse' subtype in Lauren's classification. Together, our data demonstrate the potential usefulness of our vessel subtype gene signatures in identifying tumor subtypes that may benefit from this anti-angiogenic therapy. Citation Format: Jiangang Liu, Beverly L. Falcon, Janice A. Nagy, Shou-Ching S. Jaminet, Dan Li, Thompson N. Doman, Sudhakar Chintharlapalli, Bronek Pytowski, Mark T. Uhlik, Harold Dvorak, Laura Benjamin, Anthony S. Fischl. Bioinformatics analysis of an Ad-VEGF flank angiogenesis model identifies vessel subtype gene signatures: Implications for anti-VEGF therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2359. doi:10.1158/1538-7445.AM2014-2359

Id2 and Id3 maintain the regulatory T cell pool to suppress inflammatory disease.

Regulatory T (Treg) cells suppress the development of inflammatory disease, but our knowledge of transcriptional regulators that control this function remains incomplete. Here we show that expression of Id2 and Id3 in Treg cells was required to suppress development of fatal inflammatory disease. We found that T cell antigen receptor (TCR)-driven signaling initially decreased the abundance of Id3, which led to the activation of a follicular regulatory T (TFR) cell-specific transcription signature. However, sustained lower abundance of Id2 and Id3 interfered with proper development of TFR cells. Depletion of Id2 and Id3 expression in Treg cells resulted in compromised maintenance and localization of the Treg cell population. Thus, Id2 and Id3 enforce TFR cell checkpoints and control the maintenance and homing of Treg cells.

Cell-specific translational profiling in acute kidney injury

Acute kidney injury (AKI) promotes an abrupt loss of kidney function that results in substantial morbidity and mortality. Considerable effort has gone toward identification of diagnostic biomarkers and analysis of AKI-associated molecular events; however, most studies have adopted organ-wide approaches and have not elucidated the interplay among different cell types involved in AKI pathophysiology. To better characterize AKI-associated molecular and cellular events, we developed a mouse line that enables the identification of translational profiles in specific cell types. This strategy relies on CRE recombinase-dependent activation of an EGFP-tagged L10a ribosomal protein subunit, which allows translating ribosome affinity purification (TRAP) of mRNA populations in CRE-expressing cells. Combining this mouse line with cell type-specific CRE-driver lines, we identified distinct cellular responses in an ischemia reperfusion injury (IRI) model of AKI. Twenty-four hours following IRI, distinct translational signatures were identified in the nephron, kidney interstitial cell populations, vascular endothelium, and macrophages/monocytes. Furthermore, TRAP captured known IRI-associated markers, validating this approach. Biological function annotation, canonical pathway analysis, and in situ analysis of identified response genes provided insight into cell-specific injury signatures. Our study provides a deep, cell-based view of early injury-associated molecular events in AKI and documents a versatile, genetic tool to monitor cell-specific and temporal-specific biological processes in disease modeling.

A8.16 T helper lymphocytes and monocytes as biosensors of type I interferon responses in viral infection and autoimmunity

BackgroundImmune responses in viral infections and autoimmunity are characterised by type I interferon (IFN) gene signatures detectable in peripheral blood mononuclear cells (PBMC). We have identified cell-specific IFN signatures in...

1.63 IL-7 drives the tissue architecture of the RA synovium providing a niche for B-cells to mature

BackgroundRA is a very heterogeneous disease, recently highlighted by distinct differences in the genetic contribution to APCA(+) and ACAP(–) disease suggesting two divergent pathogenic models, with different rates of progression...

Embryonic stem cell-specific signature in cervical cancer

The wide range of invasive and noninvasive lesion phenotypes associated with high-risk human papillomavirus (HR-HPV) infection in cervical cancer (CC) indicates that not only the virus but also specific cervical epithelial cells in the transformation zone (TZ), such as stem cells (SCs), play an important part in the development of cervical neoplasia. In this review, we focused in an expression signature that is specific to embryonic SCs and to poorly differentiated cervical malignant tumors and we hypothesize that this expression signature may play an important role to promote cell growth, survival, colony formation, lack of adhesion, as well as cell invasion and migration in CC.

98-P: KIR mRNA EXPRESSION IN PBMC AND ENRICHED NK CELLS ASSAYED BY QUANTITATIVE REAL-TIME PCR

To develop assays to quantify the mRNA transcribed from KIR genes utilizing a quantitative real-time PCR (qPCR) method. To study the KIR mRNA expression patterns in PBMC and enriched NK cells among healthy volunteers. PBMCs were obtained from freshly drawn whole blood (maximum 48 hours) from 14 healthy volunteers using a Ficoll method. NK cell enrichment from PBMC was performed by negative selection using a magnetic bead kit. mRNA extraction and cDNA synthesis was followed by EvaGreen based qPCR. Relative KIR mRNA expression was quantified by 2-ΔΔCt algorithm with GAPDH as reference. qPCR with a Cq ⩽ 38.0 was considered positive. 10 mRNA expression assays for the 9 KIR genes 2DL1, 2DL5A, 2DL5B, 2DS1, 2DS2, 2DS3, 2DS4 (2DS4-full length and 2DS4-truncated), 2DS5 and 3DS1 have been developed and validated. Among the 14 individuals, KIR mRNAs were detected in all samples carrying respective KIR genes and were negative for KIR gene negative samples. The mRNA expression varied among individuals. In PBMC, the relative quantification (RQ) of cDNA showed the largest variation for 2DL1 (14-fold, n = 14) and the smallest for 3DS1 (1.8-fold, n = 3) among volunteers who were positive for the respective genes. 2DL5B cDNA was not detected, as expected for this gene. More KIR cDNA was detected in the enriched NK cell fraction when compared to PBMC, with the largest difference observed for 2DL1 (NK : PBMC ratio, mean ± SD: 37.8 ± 25.0, n = 14). Development of qPCR assays with high specificity and easy applicability will be useful, because all known 15 KIR genes share > 90% identity at the genomic level. We established 10 assays for the mRNA expression of 9 KIR genes. KIR mRNA expression varied among healthy individuals carrying the same KIR genotype. Because NK cells are the predominant, but not the only source of KIR mRNA, analyzing PBMC and NK cells will allow establishing an overall and NK cell specific KIR mRNA expression signature from an individual.

Dorothy Trump

<h3>Abstract</h3> Genetic changes acquired during <i>in vitro</i> culture pose a potential risk for the successful application of stem cells in regenerative medicine. To assess mutation accumulation risks induced by culturing,...

Integrative, Multi-Platform, Whole-Genome Analyses Identify Clinically Relevant Common- and Cell-Specific Signatures in Multiple Myeloma

AbstractAbstract 3974 Background and Aim.There have been groundbreaking efforts to sequence Multiple Myeloma (MM) genomes to better define the landscape of this disease. Similar efforts to integrate multiple different 'omic' data types of clinical relevance remains a challenge. We sought to develop a systematic approach to filter the spectrum of genomic alterations through the integration of multi-platform gene expression, methylation, SNP, and microRNA microarrays with transcriptome and whole genome data, combined with clinical outcome. This approach to define clinically relevant molecular signatures in myeloma was tested using three human myeloma cell lines (HMCLs), RPMI8226, MM.1s and KMS11and compared to publically available datasets. Materials and Methods.DNA and RNA were isolated from HMCLs and applied to array-based platforms: Illumina Omni1 Quad, Illumina Human HT12v4.0 Expression BeadChip, Illumina Infinium Human Methylation 27K and 450K, and Affymetrix MicroRNA GeneChip 2.0 following manufacturers protocols. Illumina TruSeq RNA and DNA protocols were used to generate RNA-Seq and DNA-Seq (mate-pair and paired-end) libraries that were analyzed using the Illumina HiSeq2000 instrument. A novel filter was developed to identify changes specific to each HMCL and applied to each platform, separately and combined. Genes were further filtered according to their clinical relevance based on publicly available data (GSE9782) from clinical trials using quantile survival analysis. Genomic changes predicted in the array-generated data were further examined and supported by RNA- and DNA-seq data, and validated by the use of external, publicly available MM cell line (MM genome portal; Keats 2007, Cancer Cell) and patient data (GSE26849; Chapman 2011, Nature). Statistical significance of results was set at 0.01. Results.We focused our analytical pipeline development on identifying genes specifically altered in KMS11, with MM1s and RPMI8226 specific and common signatures similarly defined. Based on our cell line specific filter statistic, 120 genes were identified as having KMS11-specific significant differences: 48 expression (18 over-expressed-; 23 under-expressed), 19 methylation (10 hypermethylated; 9 hypomethylated), and 53 genes featured in regions of gain (28) or loss (15). Among the 48 genes defined as having KMS11-specific expression, 12 were identified in combination with KMS11-specific methylation changes (3 over-expressed and hypomethylated; 9 under expressed and hypermethylated). Twenty of the 120 genes showed a significant association with overall survival, 5 of which showed a significant association with treatment response. Among these genes, Epidermal growth factor receptor pathway substrate 15 (EPS15) displayed a combination of increased CpG methylation, copy number loss, and low expression. We validated EPS15 expression is lowest in KMS11 using qRT-PCR and at the protein level by western blot analysis. Western blot analysis of 10 HCMLs demonstrates that EPS15 is absent in KMS11 as well as NCI-H929 and is low in OPM-2. Interestingly all three of these lines have the t(4;14). Additionally, based on publicly available aCGH and expression data, we were able to independently validate our results in both MM cell lines and patients, with a prevalence of deletion in this region in 27% of 46 MM cell lines and 16% of 234 MM patient samples. Finally, low EPS15 expression was associated with significantly shorter overall survival (OS) among MM patients treated with Bortezomib, in contrast with significantly longer OS among Dexamethasone treated patients. Conclusion.We developed an analytical pipeline for integrative analyses to obtain molecular signatures in MM using multiple genomic data types. This approach may be useful for guiding treatment decisions, in addition to the identification of genomic changes associated with MM. We initially focused on EPS15, as one of a few filtered genes that showed clinically relevant expression differences to treatment. While this gene is located on 1p23, a deletion 'hotspot' in MM, the combination of its deletion and hyper-methylation, along with its expression difference association in response to current treatment for MM provide support for studying this gene in myeloma. Taken together, these results provide a template for large-scale, integrative informatics analyses of studies in MM as well as across many tumor types. Disclosures:No relevant conflicts of interest to declare.