Microarray-based Analysis Research Articles

Prognosis of stage III colorectal carcinomas with FOLFOX adjuvant chemotherapy can be predicted by molecular subtype.

Individualizing adjuvant chemotherapy is important in patients with advanced colorectal cancers (CRCs), and the ability to identify molecular subtypes predictive of good prognosis for stage III CRCs after adjuvant chemotherapy could be highly beneficial. We performed microarray-based gene expression analysis on 101 fresh-frozen primary samples from patients with stage III CRCs treated with FOLFOX adjuvant chemotherapy and 35 matched non-neoplastic mucosal tissues. CRC samples were classified into four molecular subtypes using nonnegative matrix factorization, and for comparison, we also grouped CRC samples using the proposed consensus molecular subtypes (CMSs). Of the 101 cases, 80 were classified into a CMS group, which shows a 79% correlation between the CMS classification and our four molecular subtypes. We found that two of our subtypes showed significantly higher disease-free survival and overall survival than the others. Group 2, in particular, which showed no disease recurrence or death, was characterized by high microsatellite instability (MSI-H, 6/21), abundant mucin production (12/21), and right-sided location (12/21); this group strongly correlated with CMS1 (microsatellite instability immune type). We further identified the molecular characteristics of each group and selected 10 potential biomarker genes from each. When these were compared to the previously reported molecular classifier genes, we found that 31 out of 40 selected genes were matched with those previously reported. Our findings indicate that molecular classification can reveal specific molecular subtypes correlating with clinicopathologic features of CRCs and can have predictive value for the prognosis for stage III CRCs with FOLFOX adjuvant chemotherapy.

Dry age-related macular degeneration like pathology in aged 5XFAD mice: Ultrastructure and microarray analysis.

Age-related macular degeneration (AMD) is a leading cause of blindness in the elderly. The two types of AMD are: dry and wet AMD. While laser-induced choroidal neovascularization has been used extensively in the studies of wet AMD, there is no established mouse model that fully recapitulates the cardinal features of dry AMD. A lack of appropriate mouse model for dry AMD has hampered the translational research on the pathogenesis of the disease and the development of therapeutic agents. We hypothesized that 5XFAD mice, an animal model for the study of Alzheimer’s disease, can be used as a mouse model for dry AMD with regard to the amyloid beta (Aβ) related pathology. In this study, the ultrastructure of the retinal pigment epithelium (RPE) of 5XFAD mice was analyzed using transmission electron microscopy. Of importance, the aged 5XFAD mice show ultrastructural changes in the RPE and Bruch’s membrane (BM) that are compatible with the cardinal features of human dry AMD, including a loss of apical microvilli and basal infolding of the RPE, increased BM thickness, basal laminar and linear deposits, and accumulation of lipofuscin granules and undigested photoreceptor outer segment-laden phagosomes. In microarray-based analysis, the RPE complex of the aged 5XFAD mice shows differential gene expression profiles consistent with dry AMD in the inflammation response, immune reaction pathway, and decreased retinol metabolism. Taken together, we suggest that aged 5XFAD mice can be used as a mouse model of dry AMD to study Aβ related pathology and develop a new therapeutic approaches.

Identification of potential target genes associated with the pathogenesis of osteoarthritis using microarray based analysis.

The aim of the present study was to investigate the molecular circuitry of osteoarthritis (OA) and identify more potential target genes for OA treatment. Microarray data of GSE32317 was downloaded from the National Center for Biotechnology Information Gene Expression Omnibus database. Differentially expressed genes (DEGs) were identified in samples of synovial membrane from patients with early stage of knee OA (OA_Early) and late stage of knee OA (OA_End) that were compared with healthy specimens. Bioinformatics analysis was applied to analyze the significant functions and pathways that were enriched by the common DEGs identified in OA_Early and OA_End samples. Furthermore, a protein‑protein interaction (PPI) network was constructed and significant modules were extracted. Transcription factors (TFs) that could regulate genes in the significant modules were identified. A total of 1,207 and 1,575 DEGs were identified in OA_Early and OA_End samples compared with healthy samples, respectively. A total of 740 genes were upregulated and 308 genes were downregulated across the OA_Early and OA_End samples. These common DEGs were enriched in different gene ontology terms and pathways, such as immune response. Angiotensinogen (AGT) and C‑X‑C motif chemokine ligand 12 (CXCL12) were identified to be hub proteins in the PPI network or in the selected module 1. In addition, the DEG lysine demethylase 2B (KDM2B) was identified as a TF that can regulate genes in the significant modules 2 and 3. In conclusion, the present study has identified AGT, CXCL12 and KDM2B as potentially essential genes associated with the pathogenesis of knee OA.

Expression of Cancer Stem Cell Markers OCT4 and CD133 in Transitional Cell Carcinomas.

Treatment failure, recurrence, and metastasis in bladder cancer are attributed to a subset of tumor cells expressing cancer stem cell (CSC) markers. This study aimed to explore the expression levels and the clinical significance of putative CSC markers OCT4 and CD133 in bladder cancer. Tissue microarray-based immunohistochemical analysis was applied to investigate the expression patterns of potential CSC markers OCT4 and CD133 in bladder cancer samples. The correlation between the expressions of each marker and clinicopathologic parameters was then analyzed. There was a significant association between OCT4 expression and the TNM stage of bladder cancer (P<0.001). Our analysis demonstrated a significant association between the intensity of staining and the presence of lamina propria and muscularis propria invasion (P=0.02 and 0.02, respectively), whereas a relative inverse correlation was found between CD133 expression with lamina propria invasion (P=0.051) and muscularis propria invasion (P=0.07). The correlation of OCT4, but not CD133, with the invasiveness of bladder cancer revealed that OCT4 can be considered as a key regulator of tumor progression, aggressive behavior, and metastasis; therefore, OCT4 can be a potential marker for targeted therapy of bladder cancer.

Microarray analysis of Arabidopsis WRKY33 mutants in response to the necrotrophic fungus Botrytis cinerea.

The WRKY33 transcription factor was reported for resistance to the necrotrophic fungus Botrytis cinerea. Using microarray-based analysis, we compared Arabidopsis WRKY33 overexpressing lines and wrky33 mutant that showed altered susceptibility to B. cinerea with their corresponding wild-type plants. In the wild-type, about 1660 genes (7% of the transcriptome) were induced and 1054 genes (5% of the transcriptome) were repressed at least twofold at early stages of inoculation with B. cinerea, confirming previous data of the contribution of these genes in B. cinerea resistance. In Arabidopsis wild-type plant infected with B. cinerea, the expressions of the differentially expressed genes encoding for proteins and metabolites involved in pathogen defense and non-defense responses, seem to be dependent on a functional WRKY33 gene. The expression profile of 12-oxo-phytodienoic acid- and phytoprostane A1-treated Arabidopsis plants in response to B. cinerea revealed that cyclopentenones can also modulate WRKY33 regulation upon inoculation with B. cinerea. These results support the role of electrophilic oxylipins in mediating plant responses to B. cinerea infection through the TGA transcription factor. Future directions toward the identification of the molecular components in cyclopentenone signaling will elucidate the novel oxylipin signal transduction pathways in plant defense.

Transcriptomic Profiling of Egg Quality in Sea Bass (Dicentrarchus labrax) Sheds Light on Genes Involved in Ubiquitination and Translation

Variable and low egg quality is a major limiting factor for the development of efficient aquaculture production. This stems from limited knowledge on the mechanisms underlying egg quality in cultured fish. Molecular analyses, such as transcriptomic studies, are valuable tools to identify the most important processes modulating egg quality. However, very few studies have been devoted to this aspect so far. Within this study, the microarray-based transcriptomic analysis of eggs (of different quality) of sea bass (Dicentrarchus labrax) was performed. An Agilent oligo microarray experiment was performed on labelled mRNA extracted from 16 batches of eggs (each batch obtained from a different female) of sea bass, in which over 24,000 published probe arrays were used. We identified 39 differentially expressed genes exhibiting a differential expression between the groups of low (fertilization rate < 60 %) and high (fertilization rate > 60 %) quality. The mRNA levels of eight genes were further analyzed by quantitative PCR. Seven genes were confirmed by qPCR to be differentially expressed in eggs of low and high quality. This study confirmed the importance of some of the genes already reported to be potential molecular quality indicators (mainly rnf213 and irf7), but we also found new genes (mainly usp5, mem-prot, plec, cenpf), which had not yet been reported to be quality-dependent in fish. These results suggest the importance of genes involved in several important processes, such as protein ubiquitination, translation, DNA repair, and cell structure and architecture; these probably being the mechanisms that contribute to egg developmental competence in sea bass.

Pyrethroid Resistance in Malaysian Populations of Dengue Vector Aedes aegypti Is Mediated by CYP9 Family of Cytochrome P450 Genes.

BackgroundDengue control and prevention rely heavily on insecticide-based interventions. However, insecticide resistance in the dengue vector Aedes aegypti, threatens the continued effectiveness of these tools. The molecular basis of the resistance remains uncharacterised in many endemic countries including Malaysia, preventing the design of evidence-based resistance management. Here, we investigated the underlying molecular basis of multiple insecticide resistance in Ae. aegypti populations across Malaysia detecting the major genes driving the metabolic resistance.Methodology/Principal FindingsGenome-wide microarray-based transcription analysis was carried out to detect the genes associated with metabolic resistance in these populations. Comparisons of the susceptible New Orleans strain to three non-exposed multiple insecticide resistant field strains; Penang, Kuala Lumpur and Kota Bharu detected 2605, 1480 and 425 differentially expressed transcripts respectively (fold-change>2 and p-value ≤ 0.05). 204 genes were commonly over-expressed with monooxygenase P450 genes (CYP9J27, CYP6CB1, CYP9J26 and CYP9M4) consistently the most up-regulated detoxification genes in all populations, indicating that they possibly play an important role in the resistance. In addition, glutathione S-transferases, carboxylesterases and other gene families commonly associated with insecticide resistance were also over-expressed. Gene Ontology (GO) enrichment analysis indicated an over-representation of GO terms linked to resistance such as monooxygenases, carboxylesterases, glutathione S-transferases and heme-binding. Polymorphism analysis of CYP9J27 sequences revealed a high level of polymorphism (except in Joho Bharu), suggesting a limited directional selection on this gene. In silico analysis of CYP9J27 activity through modelling and docking simulations suggested that this gene is involved in the multiple resistance in Malaysian populations as it is predicted to metabolise pyrethroids, DDT and bendiocarb.Conclusion/significanceThe predominant over-expression of cytochrome P450s suggests that synergist-based (PBO) control tools could be utilised to improve control of this major dengue vector across Malaysia.

Conditioned medium from persistently RSV-infected macrophages alters transcriptional profile and inflammatory response of non-infected macrophages

Cells susceptible to persistent viral infections undergo important changes in their biological functions as a consequence of the expression of viral gene products that are capable of altering the gene expression profile of the host cell. Previously, we reported that persistence of the RSV genome in a mouse macrophage cell line induces important alterations in cell homeostasis, including constitutive expression of IFN-β and other pro-inflammatory cytokines. Here, we postulated that changes in the homeostasis of non-infected macrophages could be induced by soluble factors secreted by persistently RSV- infected macrophages. To test this hypothesis, non-infected mouse macrophages were treated with conditioned medium (CM) collected from cultures of persistently RSV-infected macrophages. Total RNA was extracted and a microarray-based gene expression analysis was performed. Non-infected macrophages, treated under similar conditions with CM obtained from cultures of non-infected macrophages, were used as a control to establish differential gene expression between the two conditions. Results showed that CM from the persistently RSV-infected cultures altered expression of a total of 95 genes in non-infected macrophages, resulting in an antiviral gene-transcription profile along with inhibition of the inflammatory response, since some inflammatory genes were down-regulated, including Nlrp3 and Il-1 β, both related to the inflammasome pathway. However, down-regulation of Nlrp3 and Il-1 β was reversible upon acute RSV infection. Additionally, we observed that the inflammatory response, evaluated by secreted IL-1 β, a final product of the inflammasome activity, was enhanced during acute RSV infection in macrophages treated with CM from persistently RSV-infected cultures, compared to that in macrophages treated with the control CM. This suggests that soluble factors secreted during RSV persistence may induce an exacerbated inflammatory response in non-infected cells.

High-Throughput Sequencing of the Major Histocompatibility Complex following Targeted Sequence Capture.

The Human Major Histocompatibility Complex (MHC) is a highly polymorphic region full of immunoregulatory genes. The MHC codes for the human leukocyte antigens (HLA), proteins that present on the cellular surface and that are involved in self-non-self recognition. For matching donors and recipients for organ and stem-cell transplants it is important to know an individual's HLA haplotype determinable in this region. Now, as next-generation sequencing (NGS) platforms mature and become more and more accepted as a standard method, NGS applications have spread from research laboratories to the clinic, where they provide valid genetic insights. Here, we describe a cost-effective microarray-based sequence capture, enrichment, and NGS sequencing approach to characterize MHC haplotypes. Using this approach, ~4 MB of MHC sequence for four DNA samples (donor, recipient and the parents of the recipient) were sequenced in parallel in one NGS instrument run. We complemented this approach using microarray-based genome-wide SNP analysis. Taken together, the use of recently developed tools and protocols for sequence capture and massively parallel sequencing allows for detailed MHC analysis and donor-recipient matching.

OTX2Defines a Subgroup of Atypical Teratoid Rhabdoid Tumors With Close Relationship to Choroid Plexus Tumors

Atypical teratoid rhabdoid tumors (ATRT) are highly malignant brain tumors of early childhood that have been regarded as a homogenous entity characterized by inactivation of the SMARCB1/INI1 or SMARCA4/BRG1 genes as the only characteristic alteration. Recent studies suggest that similar to other embryonal tumors ATRT can also be divided into subgroups based on their mRNA or methylation profiles. Using microarray-based expression analysis of 12 patient ATRT specimens we demonstrated the existence of 2 subgroups of ATRT. One subgroup is characterized by high expression of OTX2, encoding a transcription factor involved in brain development. OTX2 expression was verified by immunohistochemistry and might function as a novel therapeutic target for this fatal tumor. High expression of OTX2 as well as expression of Kir7.1/KCNJ13, TRPM3 and ENPP2, which have all previously been linked to either choroid plexus epithelium or choroid plexus tumors (CPTs), suggests a close histogenetic relation of this subgroup to CPTs.

Restriction to gene flow is associated with changes in the molecular basis of pyrethroid resistance in the malaria vector Anopheles funestus

Resistance to pyrethroids, the sole insecticide class recommended for treating bed nets, threatens the control of major malaria vectors, including Anopheles funestus Effective management of resistance requires an understanding of the dynamics and mechanisms driving resistance. Here, using genome-wide transcription and genetic diversity analyses, we show that a shift in the molecular basis of pyrethroid resistance in southern African populations of this species is associated with a restricted gene flow. Across the most highly endemic and densely populated regions in Malawi, An. funestus is resistant to pyrethroids, carbamates, and organochlorides. Genome-wide microarray-based transcription analysis identified overexpression of cytochrome P450 genes as the main mechanism driving this resistance. The most up-regulated genes include cytochrome P450s (CYP) CYP6P9a, CYP6P9b and CYP6M7. However, a significant shift in the overexpression profile of these genes was detected across a south/north transect, with CYP6P9a and CYP6P9b more highly overexpressed in the southern resistance front and CYP6M7 predominant in the northern front. A genome-wide genetic structure analysis of southern African populations of An. funestus from Zambia, Malawi, and Mozambique revealed a restriction of gene flow between populations, in line with the geographical variation observed in the transcriptomic analysis. Genetic polymorphism analysis of the three key resistance genes, CYP6P9a, CYP6P9b, and CYP6M7, support barriers to gene flow that are shaping the underlying molecular basis of pyrethroid resistance across southern Africa. This barrier to gene flow is likely to impact the design and implementation of resistance management strategies in the region.

Interaction of macrophages and endometrial cells induces epithelial-mesenchymal transition-like processes in adenomyosis.

Adenomyosis is a nonneoplastic condition characterized by the benign invasion of ectopic endometrium into the myometrium. Macrophages play significant roles in epithelial-mesenchymal transition (EMT) and adenomyosis. An EMT associated with adenomyosis has been extensively studied. This study investigated the process by which the interaction of macrophages with endometrial cells induces EMT in Ishikawa cells and epithelial cells of adenomyosis. Specimens were collected after hysterectomy or resection of adenomyosis lesions from women with adenomyosis and curettage from women without adenomyosis or endometriosis. Immunohistochemistry and immunofluorescent staining demonstrated that CD68-positive macrophages aggregated in adenomyosis lesions, along with the increased protein expressions of n-cadherin, vimentin, and S100A4. By contrast, the protein expressions of e-cadherin and CK7 were decreased. After the primary endometrium cells were cocultured with THP-1-derived macrophages, the protein expression levels of n-cadherin, vimentin, and S100A4 of endometrium cells were increased, whereas the protein expression levels of e-cadherin and CK7 were decreased. The proportion of alternatively activated (M2) macrophages derived from THP-1 macrophages was also increased. The M2 macrophages elicited a bidirectional effect on Ishikawa cells by inducing EMT-like or mesenchymal-epithelial transition-like processes. The apoptotic rate of the Ishikawa cells cocultured with macrophages was increased, whereas their cell proliferation rate was decreased. Transmission electron microscopy indicated that the number of intercellular junctions of the cocultured Ishikawa cells was reduced. Microarray-based gene expression analysis revealed that transforming growth factor-β1/Smad3 and interleukin-6/JAK2/STAT3 signaling pathways were upregulated. Therefore, macrophages can induce EMT-like processes in adenomyosis and undergo polarization to M2.

Ectopic overexpression of filamin C scaffolds MEK1/2 and ERK1/2 to promote the progression of human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) invasion and metastasis are mediated by a complicated signal transduction network and downstream cytoskeletal and adhesion molecules. In this study, a microarray-based analysis revealed a dramatic increase in filamin C (FLNC), which is commonly expressed in muscle rather than in liver cells, in the two metastatic HCC cell lines MHCC97L and HCCLM3. Clinicopathological studies showed that increased FLNC expression was associated with microvascular invasion and poor prognosis. Specific hypomethylation was identified within the FLNC promoter region in HCC cell lines and patient tumor samples, which might contribute to the ectopic overexpression of FLNC. FLNC downregulation inhibited cell migration and impaired cell proliferation and promoted apoptosis. Mechanistic studies suggested that FLNC interacts with mitogen-activated extracellular signal-regulated kinase 1/2 (MEK1/2) and extracellular signal-regulated kinase 1/2 (ERK1/2) and that FLNC downregulation inhibited MEK1/2 and ERK1/2 activation. Xenographic tumor transplantation experiments in nude mice further confirmed the role of FLNC in HCC progression and metastasis. Our results reveal a novel mechanism by which the cytoskeletal protein FLNC enhances the mitogen-activated protein kinase signaling pathway during tumorigenesis.

Analysis of Leukemogenic Effects of RUNX1 and CSF3R Mutations Using Congenital Neutropenia (CN)/AML Patient-Derived Induced Pluripotent Stem Cells (iPSCs)

Severe congenital neutropenia (CN) is a pre-leukemic bone marrow failure syndrome. Recently we reported a high frequency of cooperating RUNX1 and CSF3R mutations in CN patients that developed AML or MDS. Only a combination of these two mutations induced elevated proliferation and diminished myeloid differentiation of CD34+ cells in vitro.To confirm these clinical data in an in vitro model, we generated human induced pluripotent stem cells (hiPSCs) from PBMNCs of a CN patient harbouring p.C151Y mutation in ELANE after acquisition of AML. During GCSF treatment, this patient acquired G-CSFRmutation p.Q741*, which leads to a truncated G-CSF receptor and was detected six years prior to overt AML. Three years later, he acquired an additional RUNX1 (p.R139G) mutation, which is located in the RUNT-homology domain (RHD). Subsequently, he developed AML (FAB M1) with trisomy 21. Reprogramming of PBMNCs isolated from the time-point of AML (ca. 80 % of AML blasts) resulted in the generation of hiPSCs clones harbouring either only ELANE p.C151Y mutation (CN-iPSC clone, derived from non-leukemia PBMNCs) or additional CSF3R and RUNX1 mutations and trisomy 21 (CN/AML-iPSC clone, derived from AML blasts), which was subsequently validated by Sanger sequencing and by digital PCR.These iPSCs clones have been tested for their pluripotency and self-renewal capacity. Both iPSC clones expressed the pluripotent stem cell surface markers SSEA-4 and TRA-1-60 and displayed alkaline phosphatase activity. Further they highly expressed mRNA of the pluripotent stem cell markers SOX2, ABCG2, DNMT and NANOG and were able to differentiate into all three germ layers (meso-, endo- and ectoderm).Embryoid body (EB)-based hematopoietic / neutrophilic differentiation of CN-iPS clones using serum-free APEL stem cell differentiation medium showed comparable amounts of CD34+ and CD33+ cells, but ~ 2-fold reduction of CD16+ cells, compared to healthy donor (HD) iPSCs. CN/AML-iPSCs were not able to differentiate into mature granulocytes at all and revealed 10-fold reduced counts of CD34+ and CD33+hematopoietic cells. Morphological examinations of Giemsa-stained cytospin slides confirmed these results. Additionally, CN/AML-iPSCs showed a highly reduced number of CFU-G and CFU-GM colonies in CFU-Assay.To investigate the intracellular mechanisms of leukemogenic transformation in CN, we analyzed gene expression profiles of hematopoietic cells generated from CN-iPSCs vs CN/AML-iPSCs and HD-iPSCs for various time points of differentiation in our EB based-system. Our previous microarray-based analysis of bone marrow CD33+ cells of this CN/AML patient revealed that genes overexpressed in early hematopoietic stem/progenitor cells (HSPCs) as compared to more mature progenitors, such as DNTT, BAALC, CD34, HPGDS, NPR3 and PROM1 were strongly upregulated in CN/AML blasts harbouring both RUNX1 and CSF3R mutations, as compared to the cells prior to leukemia development. Intriguingly, elevated expression of these genes was described previously in RUNX1-mutated de novo AML blasts (Mendler et al., JCO 2012). This genetic signature suggests transformation of hematopoietic progenitors carrying mutated CSF3R into more primitive hematopoietic progenitors after aquisition of RUNX1mutation. We were able to confirm markedly increase of mRNA levels of these genes in hematopoietic cells derived from CN/AML-iPSCs, as compared to CN-iPSCs.In addition, we found that hematopoietic cells of both CN-iPSCs and CN/AML-iPSCs revealed increased expression of unfolded-protein response (UPR) genes DDIT3 (CHOP), ATF4 and ATF6, as compared to HD-iPSCs. Activation of UPR in hematopoietic cells of CN-ELANEpatients has been previously described by our and other groups. CN/AML-iPSC-derived hematopoietic progenitor cells expressed RUNX1 mRNA at least two-fold higher, as compared to HD- or CN-iPSC-derived cells.In summary, we established an in vitro cellular model of leukemogenic transformation in CN patients using CN/AML-patient derived hiPSCs that confirmed clinical data of Skokowa et al. (Blood 123:2550, 2014) on a cooperative leukemogenic effect of CSF3R and RUNX1 mutations. Comprehensive analysis of hematopoiesis using this iPSCs model will give us a deeper view into this highly complex signaling network operating during leukemogenic transformation of HSCs in pre-leukemic bone marrow failure syndromes. DisclosuresNo relevant conflicts of interest to declare.

Conjunctival malignant melanoma in Denmark. Epidemiology, treatment and prognosis with special emphasis on tumorigenesis and genetic profile.

Conjunctival malignant melanoma is a rare disease associated with considerable mortality. Most published data have been based on case reports or series of referred patients. In addition, very little is known about the genetic and epigenetic profile of conjunctival melanoma. The purpose of this thesis was: To relate clinicopathological features and choice of treatment of conjunctival melanoma patients to prognosis (paper I) and to determine the incidence rate of conjunctival melanoma in Denmark (paper II). A further aim was to determine the prevalence of BRAF mutations in conjunctival melanoma, using droplet digital polymerase chain reaction (ddPCR) and immunohistochemistry (IHC), and to determine whether BRAF mutations are early events in pathogenesis and relate clinicopathological features and prognosis to BRAF mutation status (paper I and II). Finally, we wanted to identify tumour specific and prognostic microRNAs in conjunctival melanomas using a microarray-based analysis and to compare these with the microRNA expression of other melanoma subtypes (paper III). The study was based on: 139 conjunctival melanomas that had been surgically removed in Denmark over a period of 52 years (1960–2012). Conclusions: The overall incidence of conjunctival melanoma was 0.5/1 000 000/year and it increased in Denmark over 52 years. The increase was mainly caused by an increase in older patients (>65 years), bulbar lesions and lesions associated with a primary acquired melanosis with atypia (Larsen et al. 2016). Clinicopathological features significantly associated with a poor prognosis were extrabulbar location, involvement of adjacent tissue structures, tumour thickness exceeding 2 mm and local tumour recurrence. Patients undergoing incisional biopsy and/or treatment involving excision without adjuvant therapy fared worse than patients treated with excision and any type of adjuvant treatment (Larsen et al. 2015; Larsen 2016). We found that 35% (39/110) of conjunctival melanomas were BRAF mutated and the incidence of BRAF mutations was constant over time. BRAF mutation status corresponded in conjunctival melanoma and paired premalignant lesions. BRAF mutations were more frequent in males, in young patients and in tumours with a sun-exposed tumour location (bulbar conjunctiva or caruncle), with a mixed or non-pigmented colour, with absence of primary acquired melanosis and with origin in a nevus. Immunohistochemistry was able to accurately detect BRAF V600E mutations (Larsen et al. 2016). In univariate analysis, distant metastatic disease was associated with BRAF mutations; however, no prognostic associations with BRAF mutations were identified in multivariate analyses (Larsen et al. 2015, 2016). MicroRNA expression analysis revealed 24 upregulated and one downregulated microRNAs in conjunctival melanoma, several of these microRNAs have key functions in the pathogenesis and progression of cutaneous melanoma. Additionally, we identified seven upregulated microRNAs (miR-30d-5p, miR-138-5p, miR-146a-5p, miR-500a-5p, miR-501-3p, miR-501-5p and miR-502-3p) specific for stage T1 and T2 conjunctival melanoma, whose expression was associated with increased tumour thickness. No stage T3-specific microRNAs were identified. Our analysis revealed that the microRNA expression did not differ significantly between conjunctival melanoma and sinonasal or laryngeal mucosal melanoma (Larsen 2016). From our findings of a distinct pattern of BRAF mutations and differentially expressed microRNAs, conjunctival melanoma seems closely related to cutaneous and other mucosal melanomas and bears less resemblance to uveal melanomas. This means that conjunctival melanoma patients may benefit from therapies that are effective for cutaneous and mucosal melanoma. Additionally, the identification of several upregulated microRNAs may prove to be useful as future prognostic or therapeutic targets in conjunctival melanoma.

MAGE-A family expression is correlated with poor survival of patients with lung adenocarcinoma: a retrospective clinical study based on tissue microarray

ObjectivesAs the best characterised cancer/testis antigen family members, melanoma-associated antigens (MAGE) have been reported to be expressed in various malignant tumours. However, the expression pattern of MAGE-A family in lung...

Microarray-based analysis and clinical validation identify ubiquitin-conjugating enzyme E2E1 (UBE2E1) as a prognostic factor in acute myeloid leukemia.

BackgroundPrevious research suggested that single gene expression might be correlated with acute myeloid leukemia (AML) survival. Therefore, we conducted a systematical analysis for AML prognostic gene expressions.MethodsWe performed a microarray-based analysis for correlations between gene expression and adult AML overall survival (OS) using datasets GSE12417 and GSE8970. Positive findings were validated in an independent cohort of 50 newly diagnosed, non-acute promyelocytic leukemia (APL) AML patients by quantitative RT-PCR and survival analysis.ResultsMicroarray-based analysis suggested that expression of eight genes was each associated with 1-year and 3-year AML OS in both GSE12417 and GSE8970 datasets (p < 0.05). Next, we validated our findings in an independent cohort of AML samples collected in our hospital. We found that ubiquitin-conjugating enzyme E2E1 (UBE2E1) expression was adversely correlated with AML survival (p = 0.04). Multivariable analysis showed that UBE2E1 high patients had a significant shorter OS and shorter progression-free survival after adjusting other known prognostic factors (p = 0.03). At last, we found that UBE2E1 expression was negatively correlated with patients’ response to induction chemotherapy (p < 0.05).ConclusionsIn summary, we demonstrated that UBE2E1 expression was a novel prognostic factor in adult, non-APL AML patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0356-0) contains supplementary material, which is available to authorized users.

Detection of clinically relevant copy-number variants by exome sequencing in a large cohort of genetic disorders

Purpose:Copy-number variation is a common source of genomic variation and an important genetic cause of disease. Microarray-based analysis of copy-number variants (CNVs) has become a first-tier diagnostic test for patients with neurodevelopmental disorders, with a diagnostic yield of 10–20%. However, for most other genetic disorders, the role of CNVs is less clear and most diagnostic genetic studies are generally limited to the study of single-nucleotide variants (SNVs) and other small variants. With the introduction of exome and genome sequencing, it is now possible to detect both SNVs and CNVs using an exome- or genome-wide approach with a single test.Methods:We performed exome-based read-depth CNV screening on data from 2,603 patients affected by a range of genetic disorders for which exome sequencing was performed in a diagnostic setting.Results:In total, 123 clinically relevant CNVs ranging in size from 727 bp to 15.3 Mb were detected, which resulted in 51 conclusive diagnoses and an overall increase in diagnostic yield of ~2% (ranging from 0 to –5.8% per disorder).Conclusions:This study shows that CNVs play an important role in a broad range of genetic disorders and that detection via exome-based CNV profiling results in an increase in the diagnostic yield without additional testing, bringing us closer to single-test genomics.Genet Med advance online publication 27 October 2016

Development of a microarray-based assay for rapid monitoring of genetic variants of West Nile virus circulating in the United States

West Nile virus (WNV) has become endemic in the Western Hemisphere since its first introduction in the United States in 1999. An important factor associated with annual reoccurrence of WNV outbreaks in the U.S. is viral adaptation to domestic mosquitoes and birds through accumulation of spontaneous mutations in the WNV genome. Newly emerged mutations in the viral genome can potentially negatively affect the performance of existing diagnostic and screening assays and future vaccines. Therefore, the genetic monitoring of the WNV viral population during annual outbreaks is extremely important for public health and can only be achieved by application of efficient sample preparation methods followed by high throughput genetic analysis. In this study, we developed and evaluated a method for specific isolation of WNV genomic RNA from plasma samples without cultivation of the virus in cells. In combination with the microarray-based genetic analysis of the isolated WNV genomic RNA, this approach is suitable for fast, high throughput genotyping of circulating WNV genetic variants. The methods were evaluated using WNV isolates from the 1999–2012U.S. epidemics.

Transcriptomic and Metabolomic Analysis Revealed Multifaceted Effects of Phage Protein Gp70.1 on Pseudomonas aeruginosa.

The impact of phage infection on the host cell is severe. In order to take over the cellular machinery, some phage proteins were produced to shut off the host biosynthesis early in the phage infection. The discovery and identification of these phage-derived inhibitors have a significant prospect of application in antibacterial treatment. This work presented a phage protein, gp70.1, with non-specific inhibitory effects on Pseudomonas aeruginosa and Escherichia coli. Gp70.1 was encoded by early gene – orf 70.1 from P. aeruginosa phage PaP3. The P. aeruginosa with a plasmid encoding gp70.1 showed with delayed growth and had the appearance of a small colony. The combination of multifaceted analysis including microarray-based transcriptomic analysis, RT-qPCR, nuclear magnetic resonance (NMR) spectroscopy-based metabolomics and phenotype experiments were performed to investigate the effects of gp70.1 on P. aeruginosa. A total of 178 genes of P. aeruginosa mainly involved in extracellular function and metabolism were differentially expressed in the presence of gp70.1 at three examined time points. Furthermore, our results indicated that gp70.1 had an extensive impact on the extracellular phenotype of P. aeruginosa, such as motility, pyocyanin, extracellular protease, polysaccharide, and cellulase. For the metabolism of P. aeruginosa, the main effect of gp70.1 was the reduction of amino acid consumption. Finally, the RNA polymerase sigma factor RpoS was identified as a potential cellular target of gp70.1. Gp70.1 was the first bacterial inhibitor identified from Pseudomonas aeruginosa phage PaP3. It was also the first phage protein that interacted with the global regulator RpoS of bacteria. Our results indicated the potential value of gp70.1 in antibacterial applications. This study preliminarily revealed the biological function of gp70.1 and provided a reference for the study of other phage genes sharing similarities with orf70.1.