Significance Analysis Of Microarrays Research Articles

Abstract 2494: Identification of novel oncogenic targets of mutant p53 in esophageal squamous cell carcinoma

Abstract Background: Mutations in the TP53 gene are observed at a high frequency in Esophageal Squamous Cell Carcinoma (ESCC) with a majority of alterations being of the missense type affecting residues located in the region encoding the DNA binding domain. These mutations are associated with poor patient survival. However, characterization of the transcriptional networks regulated by p53 mutant proteins is limited to only a few frequent ‘hotspot' mutants, while comparatively rarer mutants have not been characterized. Objectives: Identification and characterization of differentially expressed genes associated with p53 mutation status in ESCC tumor samples. Elucidation of the mechanism of transcriptional regulation of mutant p53 target genes. Methods: A defined set of ESCC tumor samples with known p53 mutation status was selected for this study. The ability of each 'rare' p53 mutation to modulate tumor-related phenotypes was evaluated by performing phenotypic assays such as proliferation, colony formation and migration, in head and neck cancer cell-lines. Gene expression microarray analysis was performed on 36 ESCC tumors to determine putative target genes of 'rare' p53 mutant forms. Following dimension reduction, differentially expressed (putative mutant p53 target) genes were identified using Significance Analysis of Microarrays (SAM) and validated using reverse transcription quantitative PCR (RT-qPCR). Transcription induction of the putative target genes by ectopically expressed wild-type and mutant p53, in p53-null H1299 cell-line, were also evaluated by RT-qPCR. Chromatin immuno-precipitation (ChIP) assay was used to determine localization of mutant p53 proteins to chromosomal loci harboring the putative target genes. Similarly, ability to activate target promoters by mutant p53 was validated through promoter-luciferase assay. Results: Genome-wide analysis revealed 27 differentially expressed genes, of which 9 exhibited up-regulated transcript levels in p53 mutant tumors. Three putative mutant p53 target genes - C1QBP, ARF6 and TRIM23 - were selected for further analysis due to previous reports of their association with cancer. TP53 transcript levels positively correlated with transcript levels of the identified potential mutant targets. The ectopically-expressed p53 mutants - P190T and P278L, induced the increased expression of the target genes, were localized to and activated the promoters of the target genes. Further characterization of the target genes is currently underway. Conclusions: Three novel potential oncogenic targets of mutant p53 proteins were identified. The study further strengthened the heterogeneity in the functioning of different p53 mutants. Citation Format: Sara A. George, Viswakalyan Kotapalli, Raju S. Adduri, Raju Kumar, Sanjana Sarkar, Ramaswamy Pandilla, Murali D. Bashyam. Identification of novel oncogenic targets of mutant p53 in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2494.

Micrornas in Bone Marrow Mesenchymal Stromal Cells Are Differentially Expressed According to Response in Multiple Myeloma

Background: Bone marrow microenvironment plays a crucial role in the pathogenesis of multiple myeloma (MM) by supporting plasma cell growth, survival and drug resistance. These abilities have been attributed in part to the secretion of growth factors and cytokines by bone marrow mesenchymal stromal cells (BM-MSC). microRNAs (miRNAs) are promising prognostic biomarkers and therapeutic targets that can be released and transferred among MM cells and BM-MSCs as cell-cell communication. Recently, we defined a serum 14-miRNA signature which was associated with progression and complete remission (CR) after autologous stem-cell transplantation (ASCT) in patients with MM. Patients in CR showed a partial recovery of the normal serum miRNA levels and these are similar to those samples from patients with monoclonal gammopathy of undetermined significance (MGUS). Thus, these results highlight the potential value of serum miRNAs as diagnostic and prognostic biomarkers with a cell origin different from malignant plasma cells. The aim of this study was to discover whether the MSC miRNA profile varies depending on the status of the disease.Methods: Mononuclear cells from BM samples were cultured in DMEM containing 10% FBS. After a week, non-adherent cells were removed, whereas BM-MSC were selected by their adherence to the plastic and their phenotype was confirmed by multiparametric flow cytometry. We analyzed 670 microRNAs in 20 primary BM-MSC from patients with MGUS (N=4), symptomatic MM (N=8) and MM in CR (N=8). miRNAs differentially expressed were identified according to a supervised analysis using significance analysis of microarrays (SAM) and Student's t-test based on multivariate permutation (with random variance model). miRNAs differentially expressed between groups of patients were validated in an extended cohort of BM-MSC from patients (N= 82) in all the different status disease: MGUS (N=23), MM at diagnosis (N=13), relapsed MM (N=11), MM in partial response (PR) (N=13) and MM in CR (N=22), as well as in malignant plasma cells (CD38+) from those patients with symptomatic MM. In order to identify miRNA targets, we used the RmiR package to cross-correlate the miRNA expression data from the present study with our findings on the gene expression signature in 12 BM-MSCs from patients (4 MGUS, 4 symptomatic MM and 4 in CR), based on the predicted targets from TargetScan and miRBase databases.Results: In the screening phase, we identified a miRNA profile of 10 miRNAs (miR-663b, miR-654-3p, miR-206, miR-411*, miR-885-5p, miR-668, miR-638, miR-485-3p, miR-744* and miR-199a) differentially expressed between patients with symptomatic MM and MM in CR (adjusted p-value <0.0001). In the validation phase, miR-485-3p and miR-654-3p resulted differentially expressed in the three groups of patients: MGUS, symptomatic MM and patients in CR (ANOVA test: p=0.0101 and p=0.0228, respectively) (Figure 1A and 1B). The levels of these miRNAs were significantly decreased in patients with MM when compared with MGUS, and these levels seemed to recover when patients achieved CR. These two miRNAs (miR-485-3p and miR-654-3p) were also correlated with all degrees of response in MM: diagnosis, relapse, PR and CR (ANOVA test: p=0.0154 and p=0.0487, respectively). Moreover, paired cross-correlation among these two miRNAs expression with our results in mRNA gene expression profile data showed several potential gene targets with a biological interest, such as MELK and MAP2K2 (correlation index < -0.8). Finally, miR-485-3p and miR-654-3p showed a higher expression in BM-MSC than in MM CD38+ cells (Figure 1C and 1D), suggesting MSC as cell of origin for these miRNAs.Conclusion: In patients with monoclonal gammopathies, miR-485-3p and miR-654-3p expression in mesenchymal stromal cells from bone marrow is related to the status of the disease and the response to treatment in MM. The modulation of miRNAs profile in the bone marrow microenvironment around malignant plasma cells could be an important mechanism of control by cell-cell communication and the basis for potential biomarkers in serum and bone marrow. [Display omitted] DisclosuresRosinol:Celgene: Honoraria; Janssen: Honoraria.

Molecular portrait of platelets from patients with severe asthma

Asthma is a chronic inflammatory disease characterized by bronchial hyperresponsiveness, immune cell infiltration into the airways, airway remodeling and mucus overproduction. Platelets are small anucleated cells known for their role in hemostasis and thrombosis. However, they also exhibit immune and pro-inflammatory functions. Few studies suggest their involvement in the pathophysiology of asthma. The objective of our study was to analyze the transcriptomic profile of platelets from patients with severe asthma compared to non asthmatic subjects. Blood samples were collected from 5 severe asthma patients and 5 non asthmatic controls. Platelets were purified using immunomagnetic separation (Miltenyi Biotec). Total RNA was extracted and analyzed using whole genome U133 Plus 2.0 GeneChip Affymetrix microarrays. Significance analysis of microarray was used to analyze the array data with 2-fold cut-off and false discovery rate (FDR < 5%). Principal component analysis (PCA) was performed using R software. Cluster and Treeview software packages were used for the hierarchical clustering analysis. The gene ontology (GO) enrichment analysis, the biological processes and networks of differentially expressed genes were analyzed through the use of Ingenuity Pathway Analysis. The molecular signature of platelets from severe asthmatics was significantly different from the healthy subjects. Specifically, platelets from severe asthma patients were characterized by over-expression of genes related to inflammatory response and infection including CD9, CD36, CD164, SERPINB1 , PANX1 and IFITM3 . In addition, mRNA transcripts of factors controlling metabolism and transport of molecules such as phospholipase A1 ( PLAAT1 ) and leucine rich repeat ( LRRC32 ) were up-regulated in platelets from severe asthma patients. Conversely, several genes, implicated in cell death and survival ( EHMT1 , ABCC1 , HMOX1 , GSTP1 , EIF3 ), were down-regulated. Functional annotation of the differentially expressed genes revealed dysregulation of the Th1 and Th2 activation pathways ( CD28 , IL12 , TSLPR , PI3 K) that play a critical role in the adaptive immunity and therefore impact the pathophysiology of asthma. Transcriptome profiling of platelets from severe asthma patients and non-asthmatic controls allowed us to unravel the molecular basis of their special features and to determine candidate genes differentially expressed in these studied groups. This study presents a comprehensive resource to constitute a base for future investigations on the role of platelets in patients with severe asthma.

Identification of potential serum exosomal microRNAs involved in acinar-ductal metaplasia that is a precursor of pancreatic cancer associated with chronic pancreatitis.

Backgrounds:Due to difficulty in early diagnosis of chronic pancreatitis (CP), it is urgent to find novel biomarkers to detect CP. Exosomal microRNAs (Exo-miRNAs) located in the serum may be potential diagnostic and therapeutic targets for CP.Objective:To identify differentially expressed Exo-miRNAs (DE-Exo-miRNAs) in the serum of CP patients, we performed a bioinformatics analysis.Methods:The dataset GSE128508 was downloaded from the Gene Expression Omnibus (GEO) database. The analysis was carried out using BRB-ArrayTools and significance analysis of microarrays (SAM). The target genes of DE-S-Exo-miRNAs were predicted by miRWalk databases. Further gene ontology (GO) term and Kyoto Encyclopedia of Genomes (KEGG) pathway analyses were performed with plug-in ClueGO in Cytoscape software 3.7.0. Subsequently, the interaction regulatory network between encoded proteins of target genes was performed with the Search Tool for the Retrieval of Interacting Genes (STRING) database and analyzed using plug-in Molecular Complex Detection (MCODE) and cytoHubba in Cytoscape software 3.7.0.Results:We identified 227 DE-Exo-miRNAs in the serum. Further analysis using the miRWalk database identified 5164 target genes of these miRNAs. The protein–protein interaction (PPI) regulatory network of 1912 potential target genes for hub 10 up-regulated miRNAs with high degrees and one down-regulated miRNAs were constructed using the STRING database and Cytoscape software. The functional analysis using Cytoscape software tool highlighted that target genes involved in pancreatic cancer. Acinar-ductal metaplasia (ADM) in the inflammatory environment of CP is a precursor of pancreatic cancer. Subsequently, we constructed a network of target genes associated with ADM and their miRNAs.Conclusions:Exo-miRNAs in the serum as well as their target genes may be promising targets for the early diagnosis and treatment of CP. In addition, we identified potential Exo-miRNAs involved in ADM that is a precursor of pancreatic cancer associated with CP.

Analysis of immune biomarkers in cancer patients with solid tumors versus healthy subjects

Abstract Cytokines and other immune regulatory molecules are critical players in the immune response against cancer. There is growing interest in testing the potential utility of systemic immune biomarkers to track cancer progression and to use them as predictors of effective responses to cancer therapy. The central hypothesis guiding this project is that specific immune biomarkers will serve as predictors of effective vs. ineffective immunotherapy in patients with malignant diseases. The objective of this study was to establish baseline of immune markers in patients already started treatment with immunotherapy (n=10) (T), patients starting, but not yet treated (S) with immunotherapy (n=10) and subjects without diagnosed malignant disease (W) (n=10). Blood was collected and plasma was isolated and used in the biomarker (100 markers) analysis using a protein microarray method (RayBiotech). The biomarkers in the three groups were analyzed by Principal Component Analysis, heat map with clustering, and differential expression based on p value, and Significance Analysis of Microarrays (SAM). Although 15 biomarkers were significantly different between S vs. W groups, based on SAM, only seven were found differentially expressed. Similarly, although 10 biomarkers were significantly different between T vs. W groups, based on SAM, only one biomarker was found differentially expressed. Furthermore, SAM revealed that responders (n=4) vs. stable (n=5) subgroup of patients within the T group exhibited 22 differentially expressed biomarkers. Future larger studies will be needed to evaluate whether immune markers will be able to predict effective vs. ineffective responses to immunotherapy and whether they may have therapeutic potential.

Non HLA Antibodies in Serum Prior to the Onset of Chronic Lung Allograft Dysfunction

PurposeNon-human leukocyte antigen (HLA) antibodies have been associated with chronic lung allograft dysfunction (CLAD). Whether they predate CLAD is unclear. We hypothesized that patients destined to develop CLAD might develop non-HLA antibodies prior to CLAD onset.MethodsIn a single centre retrospective study, we examined 100 consecutive crossmatch negative recipients of a bilateral lung transplant between 1 January 2013 and 31 December 2014. Using a two-colour fluorescent antigen microarray with 152 unique antigens, we measured IgG and IgM autoantibodies in pre-transplant and six months post-transplant serum samples. Log2-transformed mean fluorescent intensities (MFIs) were determined for each antigen. CLAD was determined using published definitions. Significance analysis of microarrays (SAM) was used to analyze differences in antigen reactivity with a false discovery rate of 0.05. Paired and unpaired analyses were used to examine differences within and between recipients who developed CLAD within 5 years and recipients who remained CLAD free at 5 years post-transplant.ResultsSerum samples were available for 96 recipients - 35 (36.5%) developed CLAD and 61 remained CLAD-free within 5 years. Total IgM and IgG reactivity decreased in most patients post-transplant (Fig 1A), although IgM levels were stable in patients developing CLAD. Three IgM autoantibodies - including anti-club cell secretory protein (CCSP, Fig 1B) - increased in most patients post-transplant; this increase was of greater magnitude in patients later developing CLAD. Patients destined to develop CLAD also had an increase in IgG fluorescence directed at human core histones (Fig 1C).ConclusionOur results suggest that there may be important differences in non-HLA antibody reactivity that appear before the onset of CLAD. These observations, which require validation in further studies, may also indicate a specific role for non-HLA antibodies directed at club cell secretory protein and nuclear antigens in CLAD development. Non-human leukocyte antigen (HLA) antibodies have been associated with chronic lung allograft dysfunction (CLAD). Whether they predate CLAD is unclear. We hypothesized that patients destined to develop CLAD might develop non-HLA antibodies prior to CLAD onset. In a single centre retrospective study, we examined 100 consecutive crossmatch negative recipients of a bilateral lung transplant between 1 January 2013 and 31 December 2014. Using a two-colour fluorescent antigen microarray with 152 unique antigens, we measured IgG and IgM autoantibodies in pre-transplant and six months post-transplant serum samples. Log2-transformed mean fluorescent intensities (MFIs) were determined for each antigen. CLAD was determined using published definitions. Significance analysis of microarrays (SAM) was used to analyze differences in antigen reactivity with a false discovery rate of 0.05. Paired and unpaired analyses were used to examine differences within and between recipients who developed CLAD within 5 years and recipients who remained CLAD free at 5 years post-transplant. Serum samples were available for 96 recipients - 35 (36.5%) developed CLAD and 61 remained CLAD-free within 5 years. Total IgM and IgG reactivity decreased in most patients post-transplant (Fig 1A), although IgM levels were stable in patients developing CLAD. Three IgM autoantibodies - including anti-club cell secretory protein (CCSP, Fig 1B) - increased in most patients post-transplant; this increase was of greater magnitude in patients later developing CLAD. Patients destined to develop CLAD also had an increase in IgG fluorescence directed at human core histones (Fig 1C). Our results suggest that there may be important differences in non-HLA antibody reactivity that appear before the onset of CLAD. These observations, which require validation in further studies, may also indicate a specific role for non-HLA antibodies directed at club cell secretory protein and nuclear antigens in CLAD development.

Development of a Gene-Based Prediction Model for Recurrence of Colorectal Cancer Using an Ensemble Learning Algorithm.

It is difficult to determine which patients with stage I and II colorectal cancer are at high risk of recurrence, qualifying them to undergo adjuvant chemotherapy. In this study, we aimed to determine a gene signature using gene expression data that could successfully identify high risk of recurrence among stage I and II colorectal cancer patients. First, a synthetic minority oversampling technique was used to address the problem of imbalanced data due to rare recurrence events. We then applied a sequential workflow of three methods (significance analysis of microarrays, logistic regression, and recursive feature elimination) to identify genes differentially expressed between patients with and without recurrence. To stabilize the prediction algorithm, we repeated the above processes on 10 subsets by bagging the training data set and then used support vector machine methods to construct the prediction models. The final predictions were determined by majority voting. The 10 models, using 51 differentially expressed genes, successfully predicted a high risk of recurrence within 3 years in the training data set, with a sensitivity of 91.18%. For the validation data sets, the sensitivity of the prediction with samples from two other countries was 80.00% and 91.67%. These prediction models can potentially function as a tool to decide if adjuvant chemotherapy should be administered after surgery for patients with stage I and II colorectal cancer.

Abstract PS2-01: Plk1 expression &amp; efficacy of palbociclib in advanced hormonal receptor-positive breast cancer patients from PEARL study (GEICAM 2012-03)

Abstract Background: CDK 4/6 inhibitors (CDK 4/6i) with endocrine therapy (ET) combination therapy have improved outcomes in patients (pts) with hormonal receptor positive (HR+)/human epidermal growth factor receptor negative (HER2-) advanced breast cancer (ABC). However, most pts eventually develop resistance to these drugs, and one third never respond. Aside from HR positivity, predictive markers of clinical benefit from CDK 4/6i remains elusive. We aimed to identify biomarkers of response to palbociclib (PAL) and analyze potential therapeutic targets to reverse resistance. Methods: PEARL trial is a multicenter phase 3 study that assigned 601 postmenopausal HR+/HER2- ABC pts, whose disease progressed on aromatase inhibitors (AIs), to receive PAL + ET vs capecitabine (CAPE). We performed a differential gene expression analysis in pre-treatment tumors in extreme responders to PAL using the HTG EdgeSeq Oncology Biomarker Panel (HTG Molecular Diagnostics, Inc.), containing 2534 cancer related genes. Samples were subset in 2 categories: refractory (progressive disease as best response) vs sensitive (progression-free survival (PFS) within the upper quartile). Cox regression and Significance Analysis of Microarrays (SAM) analysis adjusting for multiple comparisons were performed. Results: We analyzed 455 (75.7%) pts with pre-treatment tumors available [from them, PAL + ET arm: 229 (50.3%) pts; CAPE arm: 226 (49.7%) pts]. Fifty genes (false discovery rate (FDR)&amp;lt;0.05) were differentially expressed in pts sensitive vs refractory to PAL (E2F target genes, epithelial-to-mesenchymal transition (EMT) and cell cycle genes, mainly). Unsupervised hierarchical clustering of pts based on the expression of these genes revealed two clusters. Cluster 1 is composed mostly of resistant tumors, highly proliferative (Ki67≥20%: 70%) with a great proportion of luminal B (59%) and non-luminal tumors (19%). Cluster 2 is composed of sensitive, low proliferative (Ki67&amp;lt;20%: 58%), mostly luminal A tumors (75%). There was no difference in ESR1 mutations distribution between the two clusters (Table 1). Forty genes were up-regulated and associated with resistance, including CCNE1 and PLK1 (Polo Like Kinase 1). In the whole cohort, pts with high levels (&amp;gt; median) of PLK1 (PLK1-high) treated with PAL, had a worse PFS in a multivariate model (5.7 months (m) vs 9.3 m of median PFS in PLK1-High vs -Low; HR=1.64, 95% CI (1.25-2.34), p=0.0008; adjusted model for confounders: age, site of disease, sites of metastasis, prior chemotherapy and Ki67). There were no differences in population treated with CAPE (9.9 m vs 9.4 m, PLK1-High vs -Low; HR=0.82, 95% CI (0.56-1.21), p=0.3189). In the METABRIC cohort, PLK1-High was associated with worse overall survival in HR+/HER2- BC but not in triple negative nor in HER2+ tumors. Among HR+/HER2- tumors, PLK1 expression was higher in luminal B and HER2-enriched intrinsic subtypes. We interrogated DepMap database and found that in BC cells lines there was an inverse correlation between PLK1 expression and effect on cell viability of CDK4 CRISPR knock-out (Pearson correlation r:0.54, p=0.009), but not of CDK6 knock-out. Also, HR+/HER2-/High Ki67 BC cell lines (HCC1428, EFM19 and MCF7) showed resistance to PAL on cell proliferation assays but sensitivity to the PLK1 inhibitor BI-2536. Conclusion: High expression of PLK1 is associated with intrinsic resistance to PAL and ET, this might be overcome with PLK1 inhibition. Table 1PATIENT CHARACTERISTICSCluster 1Cluster 2ALLn=57n=47n=104RespondersSensitive42 (73.68%)14 (29.79%)56 (53.85%)Refractory15 (26.32%)33 (70.21%)48 (46.15%)ESR1Mutated9 (15.79%)13 (27.66%)22 (21.15%)Wild type45 (78.95%)34 (72.34%)79 (75.96%)Unknown3 (5.26%)0 (0%)3 (2.88%)PriorQTN42 (73.68%)31 (65.96%)73 (70.19%)Y15 (26.32%)16 (34.04%)31 (29.81%)SubtypeLumA43 (75.44%)10 (21.28%)53 (50.96%)LumB14 (24.56%)28 (59.57%)42 (40.38%)Non Luminal0 (0%)9 (19.15%)9 (8.65%)MetastasisOne21 (36.84%)15 (31.91%)36 (34.62%)Multiple36 (63.16%)32 (68.09%)68 (65.38%)KI67 20%KI67&amp;lt;2033 (57.89%)7 (14.89%)40 (38.46%)KI67≥2016 (28.07%)33 (70.21%)49 (47.12%)Unknown8 (14.04%)7 (14.89%)15 (14.42%)Objective ResponseComplete1 (1.75%)0 (0%)1 (0.96%)Partial16 (28.07%)6 (12.77%)22 (21.15%)Progressive15 (26.32%)33 (70.21%)48 (46.15%)Stable25 (43.86%)8 (17.02%)33 (31.73%) Citation Format: Angel Guerrero- Zotano, Christoph Zielinski, Miguel Gil-Gil, Manuel Ruiz-Borrego, Eva M. Ciruelos, Montserrat Munoz, Begoña Bermejo, Mireia Margeli, Antonio Antón, Tibor Csöszi, Andrés García-Palomo, Ana Santaballa, Jose Luis Alonso, Antonio Fernández, Massimo Corsaro, Jesús Herranz, Paula López, Rosalia Caballero, Christiane Thallinger, Miguel Martin. Plk1 expression &amp; efficacy of palbociclib in advanced hormonal receptor-positive breast cancer patients from PEARL study (GEICAM 2012-03) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS2-01.

Abstract PS18-11: Molecular characterization of advanced breast cancer according to site of metastasis

Abstract Background: There is an increasing need in the clinic to biopsy metastatic disease in patients with advanced breast cancer (ABC). However, the microenvironment and tumor cell biology of breast cancer metastasis is largely unknown. Here, we report a molecular characterization of ABC according to the site of metastasis. Methods: RNA from 184 FFPE metastatic samples were evaluated using the nCounter BC 360 Panel, which includes the expression of 689 BC-related genes and 82 immune-related genes. PAM50 subtypes (Basal-like, HER2-enriched [HER2-E], Luminal A, Luminal B and Normal-like) were also determined. HER2 protein expression was assessed by immunohistochemistry (IHC) in 115 tumor samples, and HER2-low tumors (i.e. 1+ or 2+ and ISH-negative) were identified. Descriptive statistics, significance analysis of microarrays (using False Discovery Rate [FDR]) and logistic regressions were used to identify organ-specific gene expression profiles. Finally, we derived an organ-specific predictor of 209-genes from our cohort, and applied it to the RNAseq-based TCGA PanCancer dataset, which includes 174 glioblastomas multiforme, 424 liver hepatocellular carcinomas and 576 lung adenocarcinomas, among other cancer-types. Results: A single metastatic tumor sample from 184 individual patients with ABC was obtained from bone (18%), liver (17%), skin (15%), brain (12%), breast (13%), lymph nodes (9%), lung (7%), pleura (5%), ovary (2%), muscle (1%) and peritoneum (1%). All PAM50 subtypes were identified across the main organ sites; however, significant differences in subtype distribution were observed (p&amp;lt;0.001). Basal-like subtype was more prevalent in brain, lung and skin metastasis (FDR=0.3%). Unsupervised analysis and principal component analysis revealed brain and liver metastasis as the most distinct. Supervised analysis identified organ-specific genes independently of PAM50 subtype, i.e.: bone-specific genes (WIF1, IBSP, MMP9 and ITGB3); brain-specific genes (CRYAB, SOX10, FGF1 and CHI3L1); liver-specific genes (ALDH1A1, CYP4F3, PCK1, and SFRP2); lung-specific genes (CAV1, WNT5A, PTGS2 and IL6); skin-specific genes (KRT14, KRT5, S100A7 and SERPINB5). Among the organ-specific gene list, 15 (30%) of the 50 PAM50 genes were identified, including up-regulation of FGFR4 in liver, ESR1 in bone, ERBB2 in lung and KRT5 and KRT14 in skin. Regarding ERBB2, a high correlation between ERBB2 mRNA and HER2 IHC expression (0, 1+, 2+ and 3+) was observed (p&amp;lt;0.001). Interestingly, HER2-low disease was identified across all PAM50 subtypes and organ sites, including bone metastasis. Regarding immune-genes, all were found differentially expressed across the main organ sites (FDR&amp;lt;5%) with lung metastasis showing the highest expression (i.e. PDCD1, CD8A, GMZA, IL1B) and liver and brain metastasis the lowest. Finally, the 209-gene organ-specific predictor in PanCancer TCGA identified 96% of glioblastomas multiforme as brain, 98.6% of liver hepatocellular carcinomas as liver and 57.1% of lung adenocarcinoma as lung. Conclusions: The main sites of metastasis in ABC have unique biological features independently of tumor molecular subtype. Our results suggest that treatment strategies based on the site(s) of metastasis should be explored. Citation Format: Fara Brasó-Maristany, Laia Paré, Núria Chic, Olga Martínez-Sáez, Tomás Pascual, Meritxell Mallafré, Blanca González-Farré, Esther Sanfeliu, Débora Martínez, Patricia Galván, Belinda Salinas, Barbara Adamo, Reinaldo Moreno, Maria Vidal, Montserrat Muñoz, Aleix Prat. Molecular characterization of advanced breast cancer according to site of metastasis [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-11.

Modified Significance Analysis of Microarrays in Heterogeneous Diseases.

Significance analysis of microarrays (SAM) provides researchers with a non-parametric score for each gene based on repeated measurements. However, it may lose certain power in general statistical tests to correctly detect differentially expressed genes (DEGs) which violate homogeneity. Monte Carlo simulation shows that the “half SAM score” can maintain type I error rates of about 0.05 based on assumptions of normal and non-normal distributions. The author found 265 DEGs using the half SAM scoring, more than the 119 DEGs detected by SAM, with the false discovery rate controlled at 0.05. In conclusion, the author recommends the half SAM scoring method to detect DEGs in data that show heterogeneity.

FRZB as a key molecule in abdominal aortic aneurysm progression affecting vascular integrity.

Abdominal aortic aneurysm (AAA), when ruptured, results in high mortality. The identification of molecular pathways involved in AAA progression is required to improve AAA prognosis. The aim of the present study was to assess the key genes for the progression of AAA and their functional role. Genomic and clinical data of three independent cohorts were downloaded from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) (GSE57691, GSE7084, and GSE98278). To develop AAA diagnosis and progression-related differentially expressed genes (DEGs), we used a significance analysis of microarray (SAM). Spearman correlation test and gene set analysis were performed to identify potential enriched pathways for DEGs. Only the Frizzled-related protein (FRZB) gene and chromosome 1 open reading frame 24 (C1orf24) exhibited significant down-regulation in all analyses. With FRZB, the pathways were associated with RHO GTPase and elastin fiber formation. With C1orf24, the pathways were elastic fiber formation, extracellular matrix organization, and cell–cell communication. Since only FRZB was evolutionally conserved in the vertebrates, function of FRZB was validated using zebrafish embryos. Knockdown of frzb remarkably reduced vascular integrity in zebrafish embryos. We believe that FRZB is a key gene involved in AAA initiation and progression affecting vascular integrity.

Potential biomarkers: differentially expressed proteins of the extrinsic coagulation pathway in plasma samples from patients with depression

ABSTRACT Depression is a severe disabling psychiatric illness and the pathophysiological mechanisms remain unknown. In previous work, we found the changes in extrinsic coagulation (EC) pathway proteins in depressed patients compared with healthy subjects were significant. In this study, we screened differentially expressed proteins (DEPs) in the EC pathway, and explored the molecular mechanism by constructing a protein-protein interaction (PPI) network. The DEPs of the EC pathwaywere initially screened by isobaric tags for relative and absolute quantification (iTRAQ) in plasma samples obtained from 20 depression patients and 20 healthy controls, and were then identified by Enzyme-linked immunosorbent assays (ELISAs). Ingenuity Pathway Analysis (IPA) software was used to analyse pathway. The differentially expressed genes (DEGs) were identified by analyzing the GSE98793 microarray data from the Gene Expression Omnibus database using the Significance Analysis for Microarrays (SAM, version 4.1) statistical method. Cytoscape version 3.4.0 software was used to construct and visualize PPI networks. The results show that Fibrinogen alpha chain (FGA), Fibrinogen beta chain (FGB), Fibrinogen gamma chain (FGG) and Coagulation factor VII (FVII) were screened in the EC pathway from depression patient samples. FGA, FGB, and FGG were significantly up-regulated, and FVII was down-regulated. Thirteen DEGs related to depression and EC pathways were identified from the microarray database. Among them NF-κB Inhibitor Beta (NFKBIB) and Heat shock protein family B (small) member 1 (HSPB1) were highly correlated with EC pathway. We conclude that EC pathway is associated with depression, which provided clues for the biomarker development and the pathogenesis of depression.

Aberrantly Methylated-Differentially Expressed Genes Identify Novel Atherosclerosis Risk Subtypes.

Increasing evidence has indicated that modulation of epigenetic mechanisms, especially methylation and long-non-coding RNA (lncRNA) regulation, plays a pivotal role in the process of atherosclerosis; however, few studies focused on revealing the epigenetic-related subgroups during atherosclerotic progression using unsupervised clustering analysis. Hence, we aimed to identify the epigenetics-related differentially expressed genes associated with atherosclerosis subtypes and characterize their clinical utility in atherosclerosis. Eighty samples with expression data (GSE40231) and 49 samples with methylation data (GSE46394) from a large artery plaque were downloaded from the GEO database, and aberrantly methylated–differentially expressed (AMDE) genes were identified based on the relationship between methylation and expression. Furthermore, we conducted weighted correlation network analysis (WGCNA) and co-expression analysis to identify the core AMDE genes strongly involved in atherosclerosis. K-means clustering was used to characterize two subtypes of atherosclerosis in GSE40231, and then 29 samples were recognized as validation dataset (GSE28829). In a blood sample cohort (GSE90074), chi-square test and logistic analysis were performed to explore the clinical implication of the K-means clusters. Furthermore, significance analysis of microarrays and prediction analysis of microarrays (PAM) were applied to identify the signature AMDE genes. Moreover, the classification performance of signature AMDE gene-based classifier from PAM was validated in another blood sample cohort (GSE34822). A total of 1,569 AMDE mRNAs and eight AMDE long non-coding RNAs (lncRNAs) were identified by differential analysis. Through the WGCNA and co-expression analysis, 32 AMDE mRNAs and seven AMDE lncRNAs were identified as the core genes involved in atherosclerosis development. Functional analysis revealed that AMDE genes were strongly related to inflammation and axon guidance. In the clinical analysis, the atherosclerotic subtypes were associated with the severity of coronary artery disease and risk of adverse events. Eight genes, including PARP15, SERGEF, PDGFD, MRPL45, UBR1, STAU1, WIZ, and LSM4, were selected as the signature AMDE genes that most significantly differentiated between atherosclerotic subtypes. Ultimately, the area under the curve of signature AMDE gene-based classifier for atherosclerotic subtypes was 0.858 and 0.812 in GSE90074 and GSE34822, respectively. This study identified the AMDE genes (lncRNAs and mRNAs) that could be implemented in clinical clustering to recognize high-risk atherosclerotic patients.

Identification of common differentially expressed genes in Turner (45,X) and Klinefelter (47,XXY) syndromes using bioinformatics analysis.

BackgroundAnalysis of patients with chromosomal abnormalities, including Turner syndrome and Klinefelter syndrome, has highlighted the importance of X‐linked gene dosage as a contributing factor for disease susceptibility. Escape from X‐inactivation and X‐linked imprinting can result in transcriptional differences between normal men and women as well as in patients with sex chromosome abnormalities.ObjectiveTo identify differentially expressed genes among patients with Turner (45,X) and Klinefelter (46,XXY) syndrome using bioinformatics analysis.MethodologyTwo gene expression data sets of Turner (45,X) and Klinefelter syndrome (47,XXY) were obtained from the Gene Omnibus Expression (GEO) database of the National Center for Biotechnology Information (NCBI). Statistical analysis was performed using R Bioconductor libraries. Differentially expressed genes (DEGs) were determined using significance analysis of microarray (SAM). The functional annotation of the DEGs was performed with DAVID v6.8 (The Database for Annotation, Visualization, and Integrated Discovery).ResultsThere are no genes over‐expressed simultaneously in both diseases. However, when crossing the list of under‐expressed genes for 45,X cells and the list of over‐expressed genes for 47,XXY cells, there are 16 common genes: SLC25A6, AKAP17A, ASMTL, KDM5C, KDM6A, ATRX, CSF2RA, DHRSX, CD99, ZBED1, EIF1AX, MVB12B, SMC1A, P2RY8, DOCK7, DDX3X, eight of which are involved in the regulation of gene expression by epigenetic mechanisms, regulation of splicing processes and protein synthesis.ConclusionOf the 16 identified as under‐expressed in 45,X cells and over‐expressed in 47,XXY cells, 14 are located in X chromosome and 2 in autosomal chromosome; 8 of these genes are involved in the regulation of gene expression: 5 genes are related to epigenetic mechanisms, 2 in regulation of splicing processes, and 1 in the protein synthesis process. Our results are limited by it being the product of a bioinformatic analysis from mRNA isolated from whole blood, this makes necessary further exploration of the relationships between these genes and Turner syndrome and Klinefelter syndrome in the future.

The Possible Influence of Mediterranean Diet on Extracellular Vesicle miRNA Expression in Breast Cancer Survivors

The Mediterranean diet (MD) has been reported to have beneficial effects on breast cancer and cardiovascular diseases. Recently, microRNAs (miRNAs) have been suggested as biomarkers for the diagnosis and disease prognosis in cancer and cardiovascular diseases. We evaluated the influence of the MD on the plasma-derived extracellular vesicle miRNA signature of overweight breast cancer survivors. Sixteen participants instructed to adhere to the MD for eight weeks were included in this study. To curate differentially expressed miRNAs after MD intervention, we employed two methods: significance analysis of microarrays and DESeq2. The selected miRNAs were analyzed using ingenuity pathway analysis. After an eight-week intervention, body mass index, waist circumference, fasting glucose, fasting insulin, and homeostatic model assessment for insulin resistance were significantly improved. Expression levels of 798 miRNAs were comprehensively analyzed, and 42 extracellular vesicle miRNAs were significantly differentially regulated after the eight-week MD (36 were up and 6 were down-regulated). We also identified enriched pathways in genes regulated by differentially expressed 42 miRNAs, which include signaling associated with breast cancer, energy metabolism, glucose metabolism, and insulin. Our study indicates that extracellular vesicle miRNAs differentially expressed as a result of the MD might be involved in the mechanisms that relate to cardiometabolic risk factors in overweight breast cancer survivors.

Identification of population-level differentially expressed genes in one-phenotype data.

MotivationFor some specific tissues, such as the heart and brain, normal controls are difficult to obtain. Thus, studies with only a particular type of disease samples (one phenotype) cannot be analyzed using common methods, such as significance analysis of microarrays, edgeR and limma. The RankComp algorithm, which was mainly developed to identify individual-level differentially expressed genes (DEGs), can be applied to identify population-level DEGs for the one-phenotype data but cannot identify the dysregulation directions of DEGs.ResultsHere, we optimized the RankComp algorithm, termed PhenoComp. Compared with RankComp, PhenoComp provided the dysregulation directions of DEGs and had more robust detection power in both simulated and real one-phenotype data. Moreover, using the DEGs detected by common methods as the ‘gold standard’, the results showed that the DEGs detected by PhenoComp using only one-phenotype data were comparable to those identified by common methods using case-control samples, independent of the measurement platform. PhenoComp also exhibited good performance for weakly differential expression signal data.Availability and implementationThe PhenoComp algorithm is available on the web at https://github.com/XJJ-student/PhenoComp.Supplementary information Supplementary data are available at Bioinformatics online.

Metabolomics and hormonomics to crack the code of filbert growth.

Plants respond to changes in their environments through hormonal activation of a physiological cascade that redirects metabolic resources and growth. In filberts (Corylus sp.), chelated iron promotes the growth of new shoots but the mechanism(s) are not understood. To use untargeted metabolomics and hormonomics approaches to generate novel hypotheses for the morphoregulatory role of ferric ethylenediamine-N,N'-di-(ortho-hydroxyphenyl) acetic acid (Fe-EDDHA) in filbert shoot organogenesis in vitro. Data were generated using previously optimized standardized untargeted metabolomics protocols with time of flight mass spectrometry. Multivariate statistical tools (principal component and partial least squares discriminant analysis) did not detect significant differences. Discovery tools Significance Analysis of Microarrays (SAM), multiple linear regression analysis, Bayesian analysis, logical algorithms, machine learning, synthetic biotransformations, targeted hormonomics, and online resources including MetaboAnalyst were used. Starch/sucrose metabolism and shikimate pathway metabolites were increased. Dose dependent decreases were found in polyphenol metabolism, specifically ellagic acid and its methylated derivative 3,4,3'-tri-O-methylellagic acid. Hormonomics analysis revealed significant differences in phytohormones and their conjugates. FeEDDHA treatment reduced indole-3-acetic acid, abscisic acid, salicylic acid, jasmonic acid conjugates (JA-Trp, JA-Ile, OH-JA) and dihydrozeatinglucoside in regenerating explants. Serotonin (5HT) was decreased in FeEDDHA-treated regenerating tissues while the related metabolite melatonin was increased. Eight phenolic conjugates of 5HT and eight catabolites were affected by FeEDDHA indicating that metabolism to sequester, deactivate and metabolize 5HT was induced by Fe(III). Tryptophan was metabolized through kynurenine but not anthranilate. Seven novel hypotheses were generated to guide future studies to understand the regulatory control(s) of shoot organogenesis.

Characterization of immortalized human islet stromal cells reveals a MSC-like profile with pancreatic features

BackgroundMesenchymal stromal cells (MSCs) represent an interesting tool to improve pancreatic islet transplantation. They have immunomodulatory properties and secrete supportive proteins. However, the functional properties of MSCs vary according to many factors such as donor characteristics, tissue origin, or isolation methods. To counteract this heterogeneity, we aimed to immortalize and characterize adherent cells derived from human pancreatic islets (hISCs), using phenotypic, transcriptomic, and functional analysis.MethodsAdherent cells derived from human islets in culture were infected with a hTERT retrovirus vector and then characterized by microarray hybridization, flow cytometry analysis, and immunofluorescence assays. Osteogenic, adipogenic, and chondrogenic differentiation as well as PBMC proliferation suppression assays were used to compare the functional abilities of hISCs and MSCs. Extracellular matrix (ECM) gene expression profile analysis was performed using the SAM (Significance Analysis of Microarrays) software, and protein expression was confirmed by western blotting.ResultshISCs kept an unlimited proliferative potential. They exhibited several properties of MSCs such as CD73, CD90, and CD105 expression and differentiation capacity. From a functional point of view, hISCs inhibited the proliferation of activated peripheral blood mononuclear cells. The transcriptomic profile of hISCs highly clusterized with bone marrow (BM)-MSCs and revealed a differential enrichment of genes involved in the organization of the ECM. Indeed, the expression and secretion profiles of ECM proteins including collagens I, IV, and VI, fibronectin, and laminins, known to be expressed in abundance around and within the islets, were different between hISCs and BM-MSCs.ConclusionWe generated a new human cell line from pancreatic islets, with MSCs properties and retaining some pancreatic specificities related to the production of ECM proteins. hISCs appear as a very promising tool in islet transplantation by their availability (as a source of inexhaustible source of cells) and ability to secrete a supportive “pancreatic” microenvironment.

The implementation of drug reposition for alcoholic hepatitis based on a sub-pathway integration strategy.

BackgroundAlcoholic hepatitis (AH) is one of the most severe forms of liver disease. The therapies which are currently available are not entirely effective, and thus novel therapies are urgently needed. However, the development of these novel therapies is limited due to incomplete information about the molecular mechanisms involved in AH.MethodsThe microarray data (GSE28619) was downloaded from the Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) between the AH and the control samples were identified using the significant analysis of microarrays (SAM) method. Metabolic sub-pathways were identified using the SubpathwayMiner R package. Cell Counting Kit-8 (CCK-8) was used to evaluate the cell viability of AML-12 cells treated with different concentrations of ethanol or riboflavin. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were employed to show the hepatocyte function.ResultsA total of 1,041 genes were determined to be expressed differentially. We then identified 11 metabolic sub-pathways that could be involved in the development of AH. This was followed by a final integrated analysis of the sub-pathways involved in AH as well as the sub-pathways involved in the drug-affected cases. The final integration results led to the identification of 64 small molecular drugs. A potential novel drug (riboflavin) involved in the fatty acid metabolism pathway was identified for further investigation. Riboflavin at the 60 nM for 24 h could reverse ethanol-induced AML-12 cell injury and could markedly decrease ALT and AST activity. The decrease in the activities of these two enzymes was observed in a dose-dependent manner when it was compared to ethanol alone, which suggests that riboflavin has a protective effect against liver cell injury caused by alcoholism.ConclusionsTo summarize, the candidate agents which are identified in the present study might give practitioners insight into the development of novel therapeutic approaches for AH.

MiRNA profiling in renal carcinoma suggest the existence of a group of pro-angionenic tumors in localized clear cell renal carcinoma.

Renal cell carcinoma comprises a variety of entities, the most common being the clear-cell, papillary and chromophobe subtypes. These subtypes are related to different clinical evolution; however, most therapies have been developed for clear-cell carcinoma and there is not a specific treatment based on different subtypes. In this study, one hundred and sixty-four paraffin samples from primary nephrectomies for localized tumors were analyzed. MiRNAs were isolated and measured by microRNA arrays. Significance Analysis of Microarrays and Consensus Cluster algorithm were used to characterize different renal subtypes. The analyses showed that chromophobe renal tumors are a homogeneous group characterized by an overexpression of miR 1229, miR 10a, miR 182, miR 1208, miR 222, miR 221, miR 891b, miR 629-5p and miR 221-5p. On the other hand, clear cell renal carcinomas presented two different groups inside this histological subtype, with differences in miRNAs that regulate focal adhesion, transcription, apoptosis and angiogenesis processes. Specifically, one of the defined groups had an overexpression of proangiogenic microRNAs miR185, miR126 and miR130a. In conclusion, differences in miRNA expression profiles between histological renal subtypes were established. In addition, clear cell renal carcinomas had different expression of proangiogenic miRNAs. With the emergence of antiangiogenic drugs, these differences could be used as therapeutic targets in the future or as a selection method for tailoring personalized treatments.