Specific Gene Expression Profiles Research Articles

Specific gene expression profiles are associated with a pathologic complete response to neoadjuvant therapy in esophageal adenocarcinoma

BackgroundPredicting treatment response to chemo-radiotherapy (CRT) in esophageal cancer remains an unrealized goal despite studies linking constellations of genes to prognosis. We aimed to determine if specific expression profiles are associated with pathologic complete response (pCR) after neoadjuvant CRT. MethodsEleven genes previously associated with esophageal cancer prognosis were identified. Esophageal adenocarcinoma (EAC) patients treated with neoadjuvant CRT and esophagectomy were included. Patients were classified into two groups: pCR and no-or-incomplete response (NR). Polymerase chain reaction was used to evaluate gene expression. Omnibus testing was applied to overall gene expression differences between groups, and log-rank tests compared individual genes. ResultsEleven pCR and eighteen NR patients were analyzed. Combined expression profiles were significantly different between pCR and NR groups (p < 0.01). The gene CCL28 was over-expressed in pCR patients (Log-HR: 1.53, 95%CI: 0.46–2.59, p = 0.005), and DKK3 was under-expressed in pCR (Log-HR: −1.03 95%CI: −1.97, −0.10, p = 0.031). ConclusionEAC tumors that demonstrated a pCR have genetic profiles that are significantly different from typical NR profiles. The genes CCL28 and DKK3 are potential predictors of treatment response.

Cellular and Molecular Characterization of Microglia: A Unique Immune Cell Population.

Microglia are essential for the development and function of the adult brain. Microglia arise from erythro-myeloid precursors in the yolk sac and populate the brain rudiment early during development. Unlike monocytes that are constantly renewed from bone marrow hematopoietic stem cells throughout life, resident microglia in the healthy brain persist during adulthood via constant self-renewal. Their ontogeny, together with the absence of turnover from the periphery and the singular environment of the central nervous system, make microglia a unique cell population. Supporting this notion, recent genome-wide transcriptional studies revealed specific gene expression profiles clearly distinct from other brain and peripheral immune cells. Here, we highlight the breakthrough studies that, over the last decades, helped elucidate microglial cell identity, ontogeny, and function. We describe the main techniques that have been used for this task and outline the crucial milestones that have been achieved to reach our actual knowledge of microglia. Furthermore, we give an overview of the “microgliome” that is currently emerging thanks to the constant progress in the modern profiling techniques.

Gene regulatory network underlying the immortalization of epithelial cells

BackgroundTumorigenic transformation of human epithelial cells in vitro has been described experimentally as the potential result of spontaneous immortalization. This process is characterized by a series of cell–state transitions, in which normal epithelial cells acquire first a senescent state which is later surpassed to attain a mesenchymal stem–like phenotype with a potentially tumorigenic behavior. In this paper we aim to provide a system–level mechanistic explanation to the emergence of these cell types, and to the time–ordered transition patterns that are common to neoplasias of epithelial origin. To this end, we first integrate published functional and well–curated molecular data of the components and interactions that have been found to be involved in such cell states and transitions into a network of 41 molecular components. We then reduce this initial network by removing simple mediators (i.e., linear pathways), and formalize the resulting regulatory core into logical rules that govern the dynamics of each of the network components as a function of the states of its regulators.ResultsComputational dynamic analysis shows that our proposed Gene Regulatory Network model recovers exactly three attractors, each of them defined by a specific gene expression profile that corresponds to the epithelial, senescent, and mesenchymal stem–like cellular phenotypes, respectively. We show that although a mesenchymal stem–like state can be attained even under unperturbed physiological conditions, the likelihood of converging to this state is increased when pro–inflammatory conditions are simulated, providing a systems–level mechanistic explanation for the carcinogenic role of chronic inflammatory conditions observed in the clinic. We also found that the regulatory core yields an epigenetic landscape that restricts temporal patterns of progression between the steady states, such that recovered patterns resemble the time–ordered transitions observed during the spontaneous immortalization of epithelial cells, both in vivo and in vitro.ConclusionOur study strongly suggests that the in vitro tumorigenic transformation of epithelial cells, which strongly correlates with the patterns observed during the pathological progression of epithelial carcinogenesis in vivo, emerges from underlying regulatory networks involved in epithelial trans–differentiation during development.

Association of specific gene expression profiles with a pathologic complete response to neoadjuvant therapy in esophageal adenocarcinoma.

51 Background: Predicting prognosis in esophageal cancer remains an unrealized goal despite studies linking constellations of genes to therapeutic response. In this study, we analyzed specific predictor genes expressed in tumor specimens from our institutional repository. Our aim was to determine if specific gene expression profiles are associated with pathologic complete response (pCR) after neoadjuvant chemo-radiotherapy (CRT). Methods: We investigated eleven genes identified from prior studies (CCL28, SPARC, S100A2, SPRR3, SIRT2, NOV, PERP, PAPSS2, DCK, DKK3, ALDH1) that have significant association with esophageal cancer progression. Patients with esophageal adenocarcinoma treated with neoadjuvant CRT followed by esophagectomy at our institution between January 2011 and July 2015 were included. Quantitative real-time polymerase chain reaction was conducted on pre-treatment biopsy specimens to determine gene expression. Patients were classified into two groups: 1) pCR and, 2) no or poor response (NR) after CRT based on final pathology report. An omnibus test using Mahalanobis distance was applied to evaluate overall genetic expression differences between groups. Log-rank tests compared the differential expression of individual genes. Results: 29 patients (11 pCR and 18 NR) were analyzed. Overall, gene expression profiles were significantly different between pCR and NR patients (p &lt; 0.01). In particular, CCL28 was over-expressed in pCR (Log-HR: 1.53, 95%CI: 0.46-2.59, p = 0.005), and DKK3-was under-expressed in pCR patients (Log-HR: -1.03 95%CI: -1.97, -0.10, p = 0.031). Conclusions: Esophageal adenocarcinoma patients with a pCR after neoadjuvant therapy have genetic profiles that are significantly different from typical NR profiles. In our population, the genes CCL28 and DKK3 are potential predictors of treatment response.

Associations between gene expression profiles of invasive breast cancer and Breast Imaging Reporting and Data System MRI lexicon.

PurposeTo evaluate whether the Breast Imaging Reporting and Data System (BI-RADS) MRI lexicon could reflect the genomic information of breast cancers and to suggest intuitive imaging features as biomarkers.MethodsMatched breast MRI data from The Cancer Imaging Archive and gene expression profile from The Cancer Genome Atlas of 70 invasive breast cancers were analyzed. Magnetic resonance images were reviewed according to the BI-RADS MRI lexicon of mass morphology. The cancers were divided into 2 groups of gene clustering by gene set enrichment an alysis. Clinicopathologic and imaging characteristics were compared between the 2 groups.ResultsThe luminal subtype was predominant in the group 1 gene set and the triple-negative subtype was predominant in the group 2 gene set (55 of 56, 98.2% vs. 9 of 14, 64.3%). Internal enhancement descriptors were different between the 2 groups; heterogeneity was most frequent in group 1 (27 of 56, 48.2%) and rim enhancement was dominant in group 2 (10 of 14, 71.4%). In group 1, the gene sets related to mammary gland development were overexpressed whereas the gene sets related to mitotic cell division were overexpressed in group 2.ConclusionWe identified intuitive imaging features of breast MRI associated with distinct gene expression profiles using the standard imaging variables of BI-RADS. The internal enhancement pattern on MRI might reflect specific gene expression profiles of breast cancers, which can be recognized by visual distinction.

P1.02-074 The Gene Expression Signatures of Pulmonary Adenocarcinoma with Micropapillary Features

Invasive lung adenocarcinomas have been classified into 5 subtypes by the new WHO classification, of which the micropapillary-predominant subtype is known to have a poor prognosis. However, we previously reported that lung adenocarcinomas harboring a micropapillary component (MPC) of more than 5% showed poorer disease-free survival and overall survival in comparison to the other subtypes (JTCVS 152: 64-72; 2016), despite the MPC not being predominant. Specific biomarkers for the MPC are yet to be developed for the initial diagnosis of adenocarcinoma with an MPC. Total RNA was extracted from snap frozen archived lung adenocarcinoma samples that had been obtained by radical thoracotomy. The diagnosis of each subtype was made by independent pathologists using formalin-fixed paraffin-embedded samples. Frozen sections were made using the archived frozen sample; RNA was isolated after the confirmation of the diagnosis of each sample. RNA-sequencing was conducted using 22 adenocarcinomas and 3 normal lung tissue samples. The 22 adenocarcinomas included micropapillary (n=6), papillary (n=5), lepidic (n=5), acinar (n=3) and solid (n=3) subtypes. The reads per kilo base of exon per million mapped reads data derived from RNA-sequencing were calculated to analyze the gene expression profiles of the MPCs. The expression levels of the genes were validated by a real-time PCR. A hierarchical clustering analysis revealed a cluster of tumor samples with MPCs. Two hundred four genes were differentially expressed in adenocarcinomas that contain an MPC. A gene-ontology analysis showed the enrichment of signal-related genes, with 50 significantly upregulated genes, including BAMBI, CXCL14 and VSIG1. The results of a gene expression analysis using next generation sequencing revealed the specific gene expression profile of adenocarcinomas that contain an MPC, and several genes might be candidate diagnostic biomarkers for the subtype. In addition, these genes may play an important role in the unique characteristics of adenocarcinomas that contain an MPC.

Gene Expression Profiles and Methylation Analysis in Down Syndrome Related Acute Lymphoblastic Leukemia

BackgroundChildren with Down syndrome (DS), which caused by an extra copy of chromosome 21, are predisposed to develop acute lymphoblastic leukemia (ALL). On the other hand, in non-DS children, acquisition of chromosome 21 gain is observed in 95% of hyperdiploid (HD) ALL, which is the most common cytogenetic abnormality pattern in childhood ALL. These may suggest that gain of chromosome 21 relates molecular pathogenesis of ALL. Genetic aberrations of RUNX1 locus on chromosome 21 including iAMP21 or t(12; 21)(p13; q22)/ETV6-RUNX1 were often found in pediatric ALL. While recent studies implicated that HMGN1 or DYRK1A on chromosome 21 were associated with molecular pathogenesis of DS-ALL, it remains to be elucidated what predispose DS children to develop ALL. Compared with ALL of non-DS children, DS-ALL have uncommon genetic alterations such as mutations in JAK2 and RAS, mutations or overexpression of CRLF2. These suggest that DS-ALL may have unique biological features compared with ALL of non-DS children. Difference of biological basis between them may correlate to worse prognosis of DS-ALL. Although microarray transcript profiling provided some characteristic gene expression in DS-ALL, no study showed comprehensive transcriptome analysis in DS-ALL.PurposeThis study was conducted to elucidate comprehensive transcriptomic landscape in DS-ALL and to reveal biological features through clustering by gene expression profiling.MethodsOur cohort includes 72 pediatric B-cell precursor ALL samples (48 DS-ALL, 13 HD-ALL, and 11 euploid (EP) ALL). All HD-ALL samples gained chromosome 21. Three cases of DS-ALL and one case of EP-ALL had ETV6-RUNX1 rearrangement. We applied genome-wide analysis using whole-transcriptome sequencing (WTS) to 55 samples, Illumina 450k methylation array to 12 samples, and Illumina EPIC methylation array to 26 samples. For gene expression profiling, we conducted consensus clustering algorithms. Data from methylation arrays were normalized by beta-mixture quantile normalization and merged by common probes.ResultsOur consensus clustering analysis of gene expression stratified samples into three groups such as DS-ALL (cluster 1), DS-ALL and HD-ALL (cluster 2), and EP-ALL (cluster 3). Eight cases with DS-ALL having RAS mutations were classified into cluster 1. DYRK1A, HMGN1, ETS2 on chromosome 21 were highly expressed in cluster 1 and cluster 2 compared with cluster 3. Expression of RUNX1 was lower in cluster 1 and cluster 2 although it was not significant. In addition, FLT3, ELK1, and ETV6 were more highly expressed in cluster 1 and cluster 2 compared with cluster 3. Comparing cluster 1 with cluster 2, NRAS, MAP2K1, JUN, FOS, WT1, EPOR, and PDGFRB were highly expressed in cluster 1. Methylation analysis indicated that methylation pattern of DS-ALL was distinct from that of HD-ALL. Using methylation analysis, we detected differentially methylated regions (DMRs) between DS-ALL and HD-ALL. In DMRs of promoter-associated regions, RUNX1, IGF2BP1, and ETV6 were included. These genes were significantly hypermethylated in DS-ALL. Next, we elucidated methylation profiling of DS-ALL compared with HD-ALL and found hypermethylated genes in DS-ALL. Although these genes may participate pathogenesis of DS-ALL, we could not find any association of gene expression pattern.ConclusionWe identified specific gene expression profiling of DS-ALL and significantly upregulated genes in DS-ALL compared with HD-ALL and EP-ALL. Overexpression of these genes was characteristic gene profiling common to ALL with gaining extra chromosome 21. While ETS2, FLT3, ELK1, NRAS, MAP2K, JUN, FOS, and WT1 were highly expressed in AML, overexpression of these genes may also be related to pathogenesis of DS-ALL. In addition, DS-ALL highly expressed PDGFRB and EPOR, to which these genes are targetable by TKIs and may improve prognosis of DS-ALL. Intriguingly, RUNX1 was hypermethylated in DS-ALL compared with HD-ALL and EP-ALL, indicating that underexpression of RUNX1 was a unique gene profiling of DS-ALL and was due to hypermethylation of RUNX1. In DS patient, hyprmethylation of RUNX1 in their normal blood cells has been reported. This aberrant hypermethylation may be related to pathogenesis of DS-ALL. In this study, we unveiled gene expression profiling and methylation pattern of DS-ALL. However, to elucidate the whole picture of molecular pathogenesis of DS-ALL, we may need further analysis. DisclosuresKataoka:Yakult: Honoraria; Boehringer Ingelheim: Honoraria; Kyowa Hakko Kirin: Honoraria. Ogawa:Kan research institute: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding.

Gene Networks and Canonical Pathways Analysis in Classical Hodgkin Lymphoma Patients

Introduction: Molecular alterations involved in development of classical Hodgkin lymphoma (cHL) are only partially known. Genetic alterations in NFkB pathway and the imbalance of T regulatory (Treg) and TH17 lymphocytes has been recognized as critical pathogenetic mechanism involved in immune scape and blockade of apoptosis in Reed-Sternberg cells. Emerging evidences suggests that NFkB activation can promote lymphocyte proliferation and survival, and therapeutic inhibition of the NFkB pathway have been proposed. Recently, our group showed an increase of circulating Treg cells in patients with cHL at diagnosis, however, after therapy, elevated levels of circulating TH17 cells was observed. Increased frequencies of Treg lymphocytes in the tumor microenvironment and peripheral blood have been proposed as one of the mechanisms for this anergic state. TH17 exhibit effector functions in immune system and tumor-infiltrating TH17 cells are associated with better prognosis in human.Objectives: In this study we aimed to evaluate the immune gene expression profile in whole blood of cHL patients at diagnosis and after treatment, and evaluate pathways and gene interaction networks.Methods: This is an open multicenter study and, so far, we included 51 patients consecutively from February 2011 to November 2015. Twenty consecutively diagnosed cHL patients, with whole blood RNA extracted at diagnosis and after treatment, were recruited for this study and prospectively evaluated. All patients were HIV negative and received ABVD chemotherapy protocol and radiotherapy if necessary. The general expression of 96 messengers RNAs present in the peripheral blood and involved in immune response was performed by a customized quantitative real-time PCR array (TaqMan¨ Low Density Array). The data was normalized with B2M mRNAs levels and relative gene expression was calculated by the 2^DDCt method, considering Wilcoxon test and Benjamini-Hochberg adjustment to correct p-values. The differentially expressed genes were used to conduct functional/pathway enrichment analysis and construct gene interaction networks. Functional annotation networks were generated using Ingenuity Pathway Analysis (IPA; Ingenuity Systems) software.Results: From the 20 patients included for this study, 12 (60%) were male, 5 (31%) had Epstein Barr virus related cHL, 18 (90%) patients presented with B symptoms, 19 (95%) patients had advanced diseases at diagnosis (stage IIBX, III and IV) and 10% of patients relapsed. We considered paired samples from 15 patients before and after treatment. At diagnosis we observed that cHL patients presented higher expression of CD274 (2 fold, p=0.018), CD28 (1.5 fold, p=0.041), CTLA-4(1.5 fold, p=0.004), FAS (1.4 fold, p=0.041), ICOS (2.1 fold, p=0.015) and IL-10 (2.7 fold, p=0.002), and decreased expression of CCL2 (-2.7 fold, p=0.026), CCL5 (-1.6 fold, p=0.012), CD40 (-1.9 fold, p=0.003), CSF2 (-1.9 fold, p=0.012). We found no association between clinical and epidemiological characteristics with immune gene expression profile. We have found that after treatment, patients displayed a specific gene expression profile related to the top 5 canonical pathways summarized in following figure:We also built a biological network to investigate the connection between the differentially expressed genes and to predict the status (activation/inhibition) of the other connected genes (nodes). The in silico analysis revealed that NFkB, a node which its signaling activation is predicted to be activated before treatment and inhibited after (following figures). The network also showed different central node molecules (molecules connected with multiple other nodes from the network), that are indirectly related to Hodgkin lymphoma and should be further investigated as potential drugable targets.Conclusions: In this study, we showed that, at diagnosis, cHL patients presented an inflammatory gene expression profile in blood that changes after treatment to an effector/immunological one. NFkB is predicted to be activated before treatment and inhibited after ABVD chemotherapy and radiotherapy. This kind of system biology approach helped us to understand that cHL associated immunosuppression and the immune reconstitution after treatment maybe the key to develop new prognostic factors and treatment strategies as well as identify drugable targets. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clinical Implementation of a Testing Algorithm for the Diagnosis of Ph-like B-Cell Acute Lymphoblastic Leukemia

Introduction:Philadelphia chromosome-like (Ph-like) B acute lymphoblastic leukemia (B-ALL) is characterized by a specific gene expression profile similar to that of Ph positive B-ALL, but lacks the BCR-ABL1 fusion. Ph-like B-ALL is known to frequently carry translocations and/or point mutations in various kinases and/or the cytokine receptor CRLF2 that activate a multi-kinase cascade and is often associated with poorer clinical outcomes. Recent reports have shown that patients with Ph-like B-ALLs respond to ABL1 or JAK2 inhibitors. However, timely diagnosis of Ph-like B-ALL cases remains clinically challenging.Methods:A panel of seven fluorescence in situ hybridization (FISH) probes targeting CRLF2, ABL1, ABL2, JAK2, PDGFRB, CSF1R and EPOR were designed and validated for the detection of rearrangements in Ph-like B-ALL. Of these, FISH for CRLF2was performed on both metaphase and interphase cells and the results were correlated with levels of CRLF2 expression assessed by multiparameter flow cytometry (MFC). Additional comprehensive analysis of fusion transcripts was conducted by PCR based testing. DNA copy number analyses were assessed using SNP microarray and/or MLPA and technologies.Results:For the initial validation, CRLF2 FISH was tested in 10 relapsed and 5 de novo B-ALL cases with CRLF2 overexpression by MFC and 100% concordance was achieved. FISH validation of other probes was successful in detecting known positive rearrangements. Using this panel, we tested a cohort of 57 B-ALL cases with a median age of 31 years old (range 13-81) and unknown Ph-like status, including 14 relapsed and 43 de novo. CRLF2 FISH was positive in all 17 (29.8%) MFC CRLF2 positive cases including 3 co-existing with the BCR-ABL1 fusion, showing 100% concordance. Of the 17 CRLF2 positive cases, five of nine cases tested (55.6%) were positive for JAK2 mutation. IGH-CRLF2 fusion was the most prevalent seen in 10 (58.9%) by metaphase FISH whereas P2RY8-CRLF2 was seen in two (11.8%) cases by metaphase FISH and/or SNP array analysis. One case had a novel fusion PAX5/ZCCHC7-CRLF2 detected by metaphase FISH analysis and the remaining 4 cases showed unknown fusion partners. In 40 (70.2%) CRLF2 negativecases, five (5/57, 8.8%) were positive with our FISH panel including two with CSF1R/PDGFRB, one with JAK2 and one with EPOR rearrangements, while one with NUP214-ABL1fusion was detected by PCR.Conclusions:Using the integrated MFC, FISH and Molecular testing, we demonstrated Ph-like B-ALL in 38.6% (22/57) of unselected B-ALL cases, including 17 CRLF2, 2 CSF1R/PDGFRB, 1 JAK2, 1 EPOR and 1 ABL1 rearrangements. Based on our clinical experience, we further implemented a clinical testing algorithm (Figure 1) for the diagnosis of Ph-like B-ALL. It is noteworthy that BCR-ABL1 fusion and CRLF2 rearrangement are not mutually exclusive and all B-ALL patients should be screened for both rearrangements simultaneously. [Display omitted] DisclosuresMullighan:Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau; Loxo Oncology: Research Funding. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding. Jain:Servier: Consultancy, Honoraria; BMS: Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Celgene: Research Funding; Abbvie: Research Funding; Seattle Genetics: Research Funding; Genentech: Research Funding; Infinity: Research Funding; Novartis: Consultancy, Honoraria; Novimmune: Consultancy, Honoraria; ADC Therapeutics: Consultancy, Honoraria, Research Funding; Incyte: Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding.

The Pan Class-I PI3K Inhibitor Copanlisib Has Preclinical Activity in Mantle Cell Lymphoma, Marginal Zone Lymphoma and Chronic Lymphocytic Leukemia As Single Agent and in Combination with Other Targeted and Conventional Agents

Introduction. Copanlisib is a pan class-I PI3K inhibitor (i) with preferred activity towards PI3Kα and PI3Kδ that is currently in clinical development as single agent and in combination for patients with non-Hodgkin lymphomas. Here, we performed a screening of copanlisib as single agent and in combination with 15 other anti-cancer agents on a panel of 17 lymphoma cell lines derived from mantle cell lymphoma (MCL), marginal zone lymphoma (MZL) and chronic lymphocytic leukemia (CLL).Methods. Cell lines derived from MCL (Jeko1, Rec1, JVM2, Granta519, Maver1, Mino1, SP-49, SP-53, UPN1, Z138), MZL (Karpas1718, VL51, SSK41, ESKOL, HAIR-M, HC-1) and CLL (MEC1) were exposed to increasing doses of copanlisib alone and in combination with increasing doses of other compounds using the fixed ratio set-up. Tested compounds were approved as well as experimental inhibitors of key regulatory pathways: e.g. anti-CD20 monoclonal antibody rituximab, BTK-i ibrutinib, CDK-i roniciclib, DNA damage agent bendamustine, HDAC-i panobinostat, HDAC1/2-i romidepsin, immunomodulatory lenalidomide, JAK1/2-i ruxolitinib, MALT-i MI2, proteasome-i bortezomib. Synergy was assessed with Chou-Talalay combination index (CI): synergism (<0.9), additive (0.9-1.1), antagonism/no benefit (> 1.1). Gene expression profiling (GEP) was done using the Illumina-HumanHT-12 Expression-BeadChips and GSEA (FDR<0.25).Results. Copanlisib showed anti-tumor activity in the vast majority of cell lines with a median IC50 of 22nM (95%C.I.; 15-98) across all the cell lines, 36nM (6-175) in MZL, 22nM (95%C.I., 3-103) in MCL, 23nM in the CLL MEC1. Among all cell lines, the most active other compounds were bortezomib (IC50 5nM; 95%C.I., 5-7), romidepsin (34nM, 2-94), roniciclib (23nM, 18-29), panobinostat (161nM, 11-1263), MI2 (490nM; 224-1000) with remaining having median IC50s >500nM. Among these the only showing higher activity in one histotype was panobinostat, more active in MCL than MZL (IC50 17 vs 557nM, P 0.04). As pattern of activity across the cell lines, copanlisib was positively correlated with ibrutinib (R 0.58, P 0.001) and negatively with ruxolitinib (R -0.55, P 0.021).When copanlisib-containing combinations were tested in the 17 cell lines, different ones frequently appeared synergistic or additive: for example copanlisib plus MI2, 88% (15); plus ibrutinib, 82% (14); plus AKT-i or panobinostat, 76% (13); plus lenalidomide or BET-i, 71% (12); plus rituximab, 65% (11); plus romidepsin, 59% (10); plus roniciclib, 53% (9); plus bortezomib, 47% (8); plus bendamustine, 35% (6); plus ruxolitinib, 12% (2). For combinations with the most promising effect, baseline GEP identified gene-sets associated with different sensitivity to these combinations. High expression of genes involved in interferon signaling, oxidative phosphorylation, fatty acid metabolism, apoptosis, PI3K/AKT/mTOR and IL6/JAK/STAT signaling and low expression of genes involved in cell cycle were associated with synergism to copanlisib combinations with several compounds. Interestingly, largely the opposite was observed for the combination with others, with better synergy in cells with high expression of E2F/MYC targets and genes involved in cell cycle and low expression of transcripts involved in interferon PI3K/AKT/mTOR and IL6/JAK/STAT signaling.Conclusion. Copanlisib was active in cell lines derived from MCL, MZL and CLL. Combinations of copanlisib with a variety of additional anti-cancer compounds were synergistic and/or additive when combined in different cell lines. The combinations of the synergy profiles in the cell lines tested and the specific GEP features might predict lymphomas that could benefit from these regimens. DisclosuresJourdan:Bayer Pharma AG: Employment. Berthold:Bayer Pharma AG: Employment. Rossi:Gilead: Honoraria, Research Funding; Abbvie: Honoraria; Janseen: Honoraria. Lange:Bayer Pharma AG: Employment. Politz:Bayer Pharma AG: Employment.

RNAi-mediated suppression of dihydroflavonol 4-reductase in tobacco allows fine-tuning of flower color and flux through the flavonoid biosynthetic pathway

To examine flux regulation in the flavonoid pathway of tobacco flowers, we suppressed two genes for dihydroflavonol 4-reductase (NtDFR 1 and 2) by RNA interference (Ri)-mediated post transcriptional gene silencing in pink-flowered tobacco. Two phenotypes were observed, pale pink (DFR-Ri_PP)- and white (DFR-Ri_W)-flowered lines. The relative mRNA levels of NtDFR genes in DFR-Ri_PP and DFR-Ri_W lines were reduced by 79%–95% relative to non-transformed (NT) plants. DFR-Ri_W lines had five-fold higher levels of small interference RNAs compared to DFR-Ri_PP lines. Expression of eight structural genes in the flavonoid pathway was significantly increased in DFR-Ri_W lines but not in DFR-Ri_PP lines based on quantitative RT-PCR. Anthocyanin contents correlated with flower color, with a reduction of 72%–97% in DFR-Ri_PP and DFR-Ri_W lines. Decreases in anthocyanin in flower were proportional with reductions of proanthocyanidin content in seeds. Two pale pink lines, DFR-Ri_PP 17 and 20, with anthocyanin decreases and the lowest level of DFR gene silencing, had higher (dihydro) flavonol production than a white flowered line, DFR-Ri_W 67. This finding suggests that suppression of DFR can increase the total levels of flavonoids due to (dihydro) flavonol biosynthesis. Our observations that higher suppression of DFR had a greater influence on the expression of flavonoid biosynthetic genes demonstrates the key role of DFR in the pathway and allows selection among DFR-Ri lines for plants with specific gene expression profiles to fine-tune flux through the pathway.

Gene-expression Analysis Identifies Specific Patterns of Dysregulated Molecular Pathways and Genetic Subgroups of Human Hepatocellular Carcinoma.

Hepatocellular carcinoma comprises of a group of heterogeneous tumors of different etiologies. The multistep process of liver carcinogenesis involves various genetic and phenotypic alterations. The molecular pathways and driver mutations involved are still under investigation. DNA micorarray technology was used to identify differentially expressed genes between human hepatocarcinoma and non-tumorous liver tissues to establish a unique specific gene-expression profile independent of the underlying liver disease. The validity of this global gene-expression profile was tested for its robustness against biopsies from other liver entities (cirrhotic and non-cirrhotic liver) by diagnosing HCC in blinded samples. Most of the consistently and strongly overexpressed genes were related to cell-cycle regulation and DNA replication [27 genes, e.g. cyclin B1, karyopherin alpha 2 (KPNA2), cyclin-dependent kinase 2 (CDC2)], G-protein depending signaling [e.g. Rac GTPase activating protein 1 (RACGAP1), Rab GTPase YPT1 homolog (RAB1), and ADP-ribosylation factor-like 2 (ARL2)] and extracellular matrix re-modelling or cytoskeleton structure [22 genes, e.g. serine proteinase inhibitor 1 kazal-type (SPINK1), osteopontin (OPN), secreted protein acidic and rich in cysteine (SPARC), collagen type 1 alpha2 (COL1A2), integrin alpha6 (ITGA6), and metalloproteinase 12 (MMP12)]. Furthermore, significantly differentially expressed genes (e.g. calcium-binding proteins, G-proteins, oncofetal proteins) in relation to tumor differentiation were detected using gene-expression analysis. It is suggested that these significantly dysregulated genes are highly specific and potentially utilizable as prognostic markers and may lead to a better understanding of human hepatocarcinogenesis.

The influence of pore size and stiffness on tenocyte bioactivity and transcriptomic stability in collagen-GAG scaffolds

Orthopedic injuries, particularly those involving tendons and ligaments, are some of the most commonly treated musculoskeletal ailments, but are associated with high costs and poor outcomes. A significant barrier in the design of biomaterials for tendon tissue engineering is the rapid de-differentiation observed for primary tenocytes once removed from the tendon body. Herein, we evaluate the use of an anisotropic collagen-glycosaminoglycan (CG) scaffold as a tendon regeneration platform. We report the effects of structural properties of the scaffold (pore size, collagen fiber crosslinking density) on resultant tenocyte bioactivity, viability, and gene expression. In doing so we address a standing hypothesis that scaffold anisotropy and strut flexural rigidity (stiffness) co-regulate long-term maintenance of a tenocyte phenotype. We report changes in equine tenocyte specific gene expression profiles and bioactivity across a homologous series of anisotropic collagen scaffolds with defined changes in pore size and crosslinking density. Anisotropic scaffolds with higher crosslinking densities and smaller pore sizes were more able to resist cell-mediated contraction forces, promote increased tenocyte metabolic activity, and maintain and increase expression of tenogenic gene expression profiles. These results suggest that control over scaffold strut flexural rigidity via crosslinking and porosity provides an ideal framework to resolve structure-function maps relating the influence of scaffold anisotropy, stiffness, and nutrient biotransport on tenocyte-mediated scaffold remodeling and long-term phenotype maintenance.

Different promoter affinities account for specificity in MYC-dependent gene regulation.

Enhanced expression of the MYC transcription factor is observed in the majority of tumors. Two seemingly conflicting models have been proposed for its function: one proposes that MYC enhances expression of all genes, while the other model suggests gene-specific regulation. Here, we have explored the hypothesis that specific gene expression profiles arise since promoters differ in affinity for MYC and high-affinity promoters are fully occupied by physiological levels of MYC. We determined cellular MYC levels and used RNA- and ChIP-sequencing to correlate promoter occupancy with gene expression at different concentrations of MYC. Mathematical modeling showed that binding affinities for interactions of MYC with DNA and with core promoter-bound factors, such as WDR5, are sufficient to explain promoter occupancies observed in vivo. Importantly, promoter affinity stratifies different biological processes that are regulated by MYC, explaining why tumor-specific MYC levels induce specific gene expression programs and alter defined biological properties of cells.

Embryo quality predictive models based on cumulus cells gene expression.

Since the introduction of in vitro fertilization (IVF) in clinical practice of infertility treatment, the indicators for high quality embryos were investigated. Cumulus cells (CC) have a specific gene expression profile according to the developmental potential of the oocyte they are surrounding, and therefore, specific gene expression could be used as a biomarker. The aim of our study was to combine more than one biomarker to observe improvement in prediction value of embryo development. In this study, 58 CC samples from 17 IVF patients were analyzed. This study was approved by the Republic of Slovenia National Medical Ethics Committee. Gene expression analysis [quantitative real time polymerase chain reaction (qPCR)] for five genes, analyzed according to embryo quality level, was performed. Two prediction models were tested for embryo quality prediction: a binary logistic and a decision tree model. As the main outcome, gene expression levels for five genes were taken and the area under the curve (AUC) for two prediction models were calculated. Among tested genes, AMHR2 and LIF showed significant expression difference between high quality and low quality embryos. These two genes were used for the construction of two prediction models: the binary logistic model yielded an AUC of 0.72 ± 0.08 and the decision tree model yielded an AUC of 0.73 ± 0.03. Two different prediction models yielded similar predictive power to differentiate high and low quality embryos. In terms of eventual clinical decision making, the decision tree model resulted in easy-to-interpret rules that are highly applicable in clinical practice.

Modulation of transcriptomic signature of the infected host: a new therapeutic strategy for the management of severe viral infections? Example of the flu

RésuméCes dernières décennies ont été marquées par l’émergence ou la réémergence de virus responsables d’épidémies ou de pandémies plus ou moins sévères. Les stratégies préventives sont prises à défaut, et l’arsenal antiviral curatif est limité d’autant plus que les résistances virales peuvent apparaître rapidement. Par ailleurs, le développement de nouvelles molécules nécessite un délai incompatible avec la réponse rapide nécessaire lors d’une épidémie d’envergure ou d’une pandémie. C’est la raison pour laquelle de nouvelles approches thérapeutiques sont nécessaires. Un concept novateur est le repositionnement de molécules déjà sur le marché en exploitant leur capacité à inverser la réponse transcriptomique cellulaire de l’hôte infecté. En identifiant des molécules qui visent l’hôte et non le virus, cette stratégie permet d’avoir un large spectre d’action et d’être potentiellement actif sur de nouveaux variants. La mise en place de cette stratégie nécessite de caractériser les réponses cellulaires spécifiques de l’infection virale d’intérêt, de cribler in silico des molécules candidates, de les tester sur modèles cellulaires et animaux, avant d’envisager des essais cliniques chez l’homme. Nous présenterons cette démarche en prenant pour exemple l’infection grippale.

Three-dimensional disorganization of the cancer genome occurs coincident with long-range genetic and epigenetic alterations

A three-dimensional chromatin state underpins the structural and functional basis of the genome by bringing regulatory elements and genes into close spatial proximity to ensure proper, cell-type–specific gene expression profiles. Here, we performed Hi-C chromosome conformation capture sequencing to investigate how three-dimensional chromatin organization is disrupted in the context of copy-number variation, long-range epigenetic remodeling, and atypical gene expression programs in prostate cancer. We find that cancer cells retain the ability to segment their genomes into megabase-sized topologically associated domains (TADs); however, these domains are generally smaller due to establishment of additional domain boundaries. Interestingly, a large proportion of the new cancer-specific domain boundaries occur at regions that display copy-number variation. Notably, a common deletion on 17p13.1 in prostate cancer spanning the TP53 tumor suppressor locus results in bifurcation of a single TAD into two distinct smaller TADs. Change in domain structure is also accompanied by novel cancer-specific chromatin interactions within the TADs that are enriched at regulatory elements such as enhancers, promoters, and insulators, and associated with alterations in gene expression. We also show that differential chromatin interactions across regulatory regions occur within long-range epigenetically activated or silenced regions of concordant gene activation or repression in prostate cancer. Finally, we present a novel visualization tool that enables integrated exploration of Hi-C interaction data, the transcriptome, and epigenome. This study provides new insights into the relationship between long-range epigenetic and genomic dysregulation and changes in higher-order chromatin interactions in cancer.

Changes in plasma chemokine C-C motif ligand 2 levels during treatment with eicosapentaenoic acid predict outcome in patients undergoing surgery for colorectal cancer liver metastasis.

The mechanism of the anti-colorectal cancer (CRC) activity of the omega-3 fatty acid eicosapentaenoic acid (EPA) is not understood. We tested the hypothesis that EPA reduces expression of chemokine C-C motif ligand 2 (CCL2), a pro-inflammatory chemokine with known roles in metastasis.We measured CCL2 in clinical samples from a randomized trial of EPA in patients undergoing liver surgery for CRC liver metastasis (LM) and preclinical models. Genome-wide transcriptional profiling of tumors from EPA-treated patients was performed.EPA decreased CCL2 synthesis by CRC cells in a dose-dependent manner. CCL2 was localized to malignant epithelial cells in human CRCLM. EPA did not reduce CCL2 content in human or mouse tumors compare to control. However, EPA treatment was associated with decreased plasma CCL2 levels compared with controls (P=0.04). Reduction in plasma CCL2 following EPA treatment predicted improved disease-free survival (HR 0.32; P=0.003). Lack of ‘CCL2 response’ was associated with a specific CRCLM gene expression signature.In conclusion, reduction in plasma CCL2 in patients with CRCLM treated with EPA predicts better clinical outcome and a specific tumor gene expression profile. Further work is needed to validate CCL2 as a therapeutic response biomarker for omega-3 fatty acid treatment of CRC patients.

VEGF-C and TGF-β reciprocally regulate mesenchymal stem cell commitment to differentiation into lymphatic endothelial or osteoblastic phenotypes.

The direction of mesenchymal stem cell (MSC) differentiation is regulated by stimulation with various growth factors and cytokines. We recently established MSC lines, [transforming growth factor-β (TGF-β)-responsive SG-2 cells, bone morphogenetic protein (BMP)-responsive SG-3 cells, and TGF-β/BMP-non-responsive SG-5 cells], derived from the bone marrow of green fluorescent protein-transgenic mice. In this study, to compare gene expression profiles in these MSC lines, we used DNA microarray analysis to characterize the specific gene expression profiles observed in the TGF-β-responsive SG-2 cells. Among the genes that were highly expressed in the SG-2 cells, we focused on vascular endothelial growth factor (VEGF) receptor 3 (VEGFR3), the gene product of FMS-like tyrosine kinase 4 (Flt4). We found that VEGF-C, a specific ligand of VEGFR3, significantly induced the cell proliferative activity, migratory ability (as shown by Transwell migration assay), as well as the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 in the SG-2 cells. Additionally, VEGF-C significantly increased the expression of prospero homeobox 1 (Prox1) and lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1), which are lymphatic endothelial cell markers, and decreased the expression of osteogenic differentiation marker genes in these cells. By contrast, TGF-β significantly increased the expression of early-phase osteogenic differentiation marker genes in the SG-2 cells and markedly decreased the expression of lymphatic endothelial cell markers. The findings of our study strongly suggest the following: i) that VEGF-C promotes the proliferative activity and migratory ability of MSCs; and ii) VEGF-C and TGF-β reciprocally regulate MSC commitment to differentiation into lymphatic endothelial or osteoblastic phenotypes, respectively. Our findings provide new insight into the molecular mechanisms underlying the regenerative ability of MSCs.

Enhancement of chondrogenic differentiation potential of equine adiposetissue-derived mesenchymal stem cells using TGF-β3 and BMP-6

This study was designed to evaluate the in vitro chondrogenic differentiation potential of equine adipose tissue-derived mesenchymal stem cells (AT-MSCs) in response to different culture conditions. Fat tissue cells after collection were cultured in optimized conditions until passage 3 (P3). P3 cells were cultured as micropellets under different chondrogenic conditions for 21 days in 5 groups: basic medium as a control, basic chondrogenic medium (BCM), BCM supplemented with transforming growth factor beta 3 (TGF-β3; 10 ng/mL), BCM supplemented with bone morphogenetic protein 6 (BMP-6; 10 ng/mL), and BCM supplemented with both TGF-β3 and BMP-6. The growth rate of pellets was measured and differentiation progression was assessed by specific stainings and gene expression profiling. Results showed that the largest pellets of cells belonged to the group containing both growth factors. All treated cells showed different levels of chondrogenic differentiation compared to the control group (P < 0.05). The differentiation score in groups containing TGF-β3 and both growth factors was significantly greater than that in other groups. Moreover, the expression of aggrecan, as a cartilage-specific gene, was confirmed in all groups expect the control group. Taken together, these results indicate that the addition of both TGF-β3 and BMP-6 to BCM potentiates the chondrogenic differentiation of equine AT-MSCs.