Significance Analysis Of Microarrays Research Articles

Metabolic profiling reveals metabolic features of consolidation therapy in pediatric acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) and its treatment continue to pose substantial risks. To understand ALL more deeply, the metabolome in fasting plasma of 27 ALL patients before and after high-dose methotrexate therapies (consolidation therapy) including methotrexate and 6-mercaptopurine (6-MP) was investigated. Plasma metabolites were analyzed using liquid chromatography–tandem mass spectrometry (LC–MS). Orthogonal projections to latent structures discriminant analysis and significance analysis of microarrays were used to evaluate the metabolic changes. Pathway enrichment and co-expression network analyses were performed to identify clusters of molecules, and 2826 metabolites were identified. Among them, 38 metabolites were identified by univariate analysis, and 7 metabolites that were altered by conditioning therapy were identified by multivariate analysis. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used for pathway enrichment analysis. Among the enriched KEGG pathways, the 3 significantly altered metabolic pathways were pyrimidine metabolism; phenylalanine, tyrosine, and tryptophan biosynthesis; and phenylalanine metabolism. In addition, L-phenylalanine was significantly correlated with blood urea nitrogen (BUN), and palmitoylcarnitine was correlated with aspartate aminotransferase (AST). In summary, consolidation therapy significantly affected pyrimidine- and phenylalanine-associated metabolic pathways in pediatric ALL patients. These findings may provide an insight into the role of metabolic profiling in consolidation treatment and as a potential for pediatric ALL patients.

Mass spectrometry-based proteomics analysis of human globus pallidus from progressive supranuclear palsy patients discovers multiple disease pathways.

Progressive supranuclear palsy (PSP) is a neurodegenerative disorder clinically characterized by progressive postural instability, supranuclear gaze palsy, parkinsonism, and cognitive decline caused by degeneration in specific areas of the brain including globus pallidus (GP), substantia nigra, and subthalamic nucleus. However, the pathogenetic mechanism of PSP remains unclear to date.Unbiased global proteome analysis of patients' brain samples is an important step toward understanding PSP pathogenesis, as proteins serve as workhorses and building blocks of the cell. In this study, we conducted unbiased mass spectrometry-based global proteome analysis of GP samples from 15 PSP patients, 15 Parkinson disease (PD) patients, and 15 healthy control (HC) individuals. To analyze 45 samples, we conducted 5 batches of 11-plex isobaric tandem mass tag (TMT)-based multiplexing experiments. The identified proteins were subjected to statistical analysis, such as a permutation-based statistical analysis in the significance analysis of microarray (SAM) method and bootstrap receiver operating characteristic curve (ROC)-based statistical analysis. Subsequently, we conducted bioinformatics analyses using gene set enrichment analysis, Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) protein-protein interaction (PPI) analysis, and weighted gene co-expression network analysis (WGCNA). We have identified 10,231 proteins with ∼1,000 differentially expressed proteins. The gene set enrichment analysis results showed that the PD pathway was the most highly enriched, followed by pathways for oxidative phosphorylation, Alzheimer disease, Huntington disease, and non-alcoholic fatty liver disease (NAFLD) when PSP was compared to HC or PD. Most of the proteins enriched in the gene set enrichment analysis were mitochondrial proteins such as cytochrome c oxidase, NADH dehydrogenase, acyl carrier protein, succinate dehydrogenase, ADP/ATP translocase, cytochrome b-c1 complex, and/or ATP synthase. Strikingly, all of the enriched mitochondrial proteins in the PD pathway were downregulated in PSP compared to both HC and PD. The subsequent STRING PPI analysis and the WGCNA further supported that the mitochondrial proteins were the most highly enriched in PSP. Our study showed that the mitochondrial respiratory electron transport chain complex was the key proteins that were dysregulated in GP of PSP, suggesting that the mitochondrial respiratory electron transport chain complex could potentially be involved in the pathogenesis of PSP. This is the first global proteome analysis of human GP from PSP patients, and this study paves the way to understanding the mechanistic pathogenesis of PSP.

Prioritizing key synergistic circulating microRNAs for the early diagnosis of biliary tract cancer.

Biliary tract cancer (BTC) is a highly aggressive malignant tumor. Serum microRNAs (ser-miRNAs) serve as noninvasive biomarkers to identify high risk individuals, thereby facilitating the design of precision therapies. The study is to prioritize key synergistic ser-miRNAs for the diagnosis of early BTC. Sampling technology, significant analysis of microarrays, Pearson Correlation Coefficients, t-test, decision tree, and entropy weight were integrated to develop a global optimization algorithm of decision forest. The source code is available at https://github.com/SuFei-lab/GOADF.git. Four key synergistic ser-miRNAs were prioritized and the synergistic classification performance was better than the single miRNA’ s. In the internal feature evaluation dataset, the area under the receiver operating characteristic curve (AUC) for each single miRNA was 0.8413 (hsa-let-7c-5p), 0.7143 (hsa-miR-16-5p), 0.8571 (hsa-miR-17-5p), and 0.9365 (hsa-miR-26a-5p), respectively, whereas the synergistic AUC value increased to 1.0000. In the internal test dataset, the single AUC was 0.6500, 0.5125, 0.6750, and 0.7500, whereas the synergistic AUC increased to 0.8375. In the independent test dataset, the single AUC was 0.7280, 0.8313, 0.8957, and 0.8303, and the synergistic AUC was 0.9110 for discriminating between BTC patients and healthy controls. The AUC for discriminating BTC from pancreatic cancer was 0.9000. Hsa-miR-26a-5p was a predictor of prognosis, patients with high expression had shorter survival than those with low expression. In conclusion, hsa-let-7c-5p, hsa-miR-16-5p, hsa-miR-17-5p, and hsa-miR-26a-5p may act as key synergistic biomarkers and provide important molecular mechanisms that contribute to pathogenesis of BTC.

Gene expression profile of human colorectal cancer identified NKTR as a biomarker for liver metastasis.

Objective: Liver metastasis is one of the prognostic factors of colorectal cancer (CRC). The aim of this study is to identify biomarkers that facilitate easier detection of liver metastasis.Methods: Significance Analysis of Microarrays (SAM) and Array Data Analyzer (ADA) were applied used for the analysis of differentially differently expressed mRNAs. mRNA expression was verified by quantitative real-timer reverse transcriptiontase polymerase chain reaction (qRT-PCR). Immunohistochemistry were was used to show natural killer-tumor recognition (NKTR) expression in CRC. NKTR-knockdown CRC cells were constructed obtained by using short hairpin RNA (shRNA). Followed by CCK-8 assay, plate colony formation test, and transwell assay were used to evaluate the influence of NKTR on cell proliferation, migration, and invasion in vitro.Results: SAM yielded showed 256 up-regulated and 224 down-regulated differentially differently expressed genes. Seven genes were identified by using ADA, tools and four genes were verified by using qRT-PCR. Three genes (metastasis associated lung adenocarcinoma transcript 1 (MALAT1), nuclear factor I/B (NKTR), and nuclear factor I/B (NFIB)) showed a statistically significant considerabley difference between CRC with and liver metastasis and CRC without liver metastasis. Immunohistochemical analysis showed that NKTR expression was much lower in primary CRC with liver metastasis than that in primary CRC without liver metastasis. The NKTR protein plays a role in the lytic function of natural killer (NK) cells and it has been rarely studied in the CRC. The down-regulation of NKTR by shRNA interference in CRC cells increased cell proliferation, migration, and invasion in vitro.

Deriving prognostic significance from a molecular subtype model of laryngeal carcinoma.

This study explored whether laryngeal carcinoma could be divided into different subtypes based on molecular differences using a molecular subtype-prediction model. We extracted data from the Cancer Genome Atlas and Gene Expression Omnibus databases and then performed unsupervised cluster analysis to identify discrete molecular subtypes of laryngeal carcinoma. Significance analysis of microarrays was performed to detect differentially expressed genes for each subtype, and gene set enrichment analysis and the GenCliP3 software were used to label gene functions and identify key pathways. We categorized 126 patients into C1 and C2 molecular subtypes associated with pathologic grade. The C2 subtype appeared more aggressive, with a worse prognosis. The most significant enrichment pathway of the C2 subtype was the Hedgehog pathway, and GLI1 was a core gene. Laryngeal carcinoma can be divided into two subtypes based on differences in molecular expression, which could identify key molecules associated with prognosis.

An 11-gene expression signature related to tumorigenesis and immunosuppression in primary cutaneous melanoma predicts sentinel lymph node metastatic status.

e21579 Background: A biomarker derived from the primary melanoma tumor to predict regional sentinel lymph node (SLN) metastatic status can be valuable in guiding the decision making in planning the SLN surgical procedure for candidate patients. Methods: Gene expression profiling was performed on primary cutaneous melanoma tumor biopsies of 49 (24 known SLN+, 25 SLN-) patients (T3a/b, T4a/b) who underwent SLN for staging using transcriptome profiling analysis. U133A 2.0 Affymetrix gene chips were used. Significance Analysis of Microarrays (SAM) was used to test the association between gene expression level of the primary tumor and SLN status. Genes with fold change > 1.5 and q value < 0.05 were considered differentially expressed. Pathway analysis was performed using Ingenuity Pathway Analysis. Benjamini and Hochberg method was used to adjust for multiple testing in pathway analysis. All statistical analyses were implemented in R. Results: A total of 49 patients with primary cutaneous melanoma were studied, of which, 24 were diagnosed as SLN positive and 25 as SLN negative by routine H&E and immunohistochemistry. Using SLN metastatic status as the outcome, a univariate logistic regression model was fitted with individual probe sets. A total of 251 probe sets were filtered and 11 probe sets were considered as differentially expressed (DE) between SLN-ve and SLN+ve groups, with the selection criterion set at Benjamini- Hochberg method -adjusted p -value below 0.05 and the absolute log fold change above 0.5. As each of the 11 probe sets was matched to a unique known gene, an 11 gene signature was derived and among them, the expression level of 7 genes was significantly reduced in the SLN+ve group compared to the SLN-ve group, while 4 genes were overexpressed in the SLN+ve group. Integrative and interactive heatmaps were produced from hierarchical cluster analysis to show the 11 differentially expressed genes. Selected genes were found to be uniformly related to tumorigenesis, malignant progression, DNA repair, cell cycle regulation, chemoresistance, immunosuppression and/or involvement in multiple cancer-related pathways. Several of the selected genes were previously shown to be prognostic in various malignancies. Conclusions: We present a unique 11-gene expression signature derived from primary melanoma tumor biopsy samples and related to tumorigenesis and immunosuppression that may be useful for the prognostic stratification of melanoma patients. This may allow the optimal selection of patients for SLN biopsy.

Anti-inflammatory effects of recreational marijuana in virally suppressed youth with HIV-1 are reversed by use of tobacco products in combination with marijuana

BackgroundMarijuana’s putative anti-inflammatory properties may benefit HIV-associated comorbidities. How recreational marijuana use affects gene expression in peripheral blood cells (PBC) among youth with HIV-1 (YWH) is unknown.ApproachYWH with defined substance use (n = 54) receiving similar antiretroviral therapy (ART) were assigned to one of four analysis groups: YWH with detectable plasma HIV-1 (> 50 RNA copies/ml) who did not use substances (H+V+S−), and YWH with undetectable plasma HIV-1 who did not use substances (H+V−S−), or used marijuana alone (H+V−S+[M]), or marijuana in combination with tobacco (H+V−S+[M/T]). Non-substance using youth without HIV infection (H−S−, n = 25) provided a reference group. PBC mRNA was profiled by Affymetrix GeneChip Human Genome U133 Plus 2.0 Array. Differentially expressed genes (DEG) within outcome groups were identified by Significance Analysis of Microarrays and used for Hierarchical Clustering, Principal Component Analysis, and Ingenuity Pathways Analysis.ResultsHIV-1 replication resulted in > 3000 DEG involving 27 perturbed pathways. Viral suppression reduced DEG to 313, normalized all 27 pathways, and down-regulated two additional pathways, while marijuana use among virally suppressed YWH resulted in 434 DEG and no perturbed pathways. Relative to H+V−S−, multiple DEG normalized in H+V−S+[M]. In contrast, H+V−S+[M/T] had 1140 DEG and 10 dysregulated pathways, including multiple proinflammatory genes and six pathways shared by H+V+S−.ConclusionsYWH receiving ART display unique transcriptome bioprofiles based on viral replication and substance use. In the context of HIV suppression, marijuana use, alone or combined with tobacco, has opposing effects on inflammatory gene expression.

Novel serological biomarker panel using protein microarray can distinguish active TB from latent TB infection

BackgroundRapid laboratory technologies which can effectively distinguish active tuberculosis (ATB) from controls and latent tuberculosis infection (LTBI) are lacked.The objective of this study is to explore MTB biomarkers in serum that can distinguish ATB from LTBI. MethodsWe constructed a tuberculosis protein microarray containing 64 MTB associated antigens. We then used this microarray to screen 180 serum samples, from patients with ATB and LTBI, and healthy volunteer controls. Both SAM (Significance analysis of microarrays) and ROC curve analysis were used to identify the differentially recognized biomarkers between groups. Extra 300 serum samples from patients with ATB and LTBI, and healthy volunteer controls were employed to validate the identified biomarkers using ELISA-based method. ResultsAccording to the results, the best biomarker combinations of 4 proteins (Rv1860, RV3881c, Rv2031c and Rv3803c) were selected. The biomarker panel containing these 4 proteins has reached a sensitivity of 93.3% and specificity of 97.7% for distinguishing ATB from LTBI, and a sensitivity of 86% and specificity of 97.6% for distinguishing ATB from HC. ConclusionThe biomarker combination in this study has high sensitivity and specificity in distinguishing ATB from LTBI, suggesting it is worthy for further validation in more clinical samples.

IgM Antibodies to Tropoelastin in Serum Prior to the Onset of Chronic Allograft Dysfunction

<h3>Purpose</h3> Non- Human Leukocyte Antigen (non-HLA) antibodies have been identified as a risk factor for chronic allograft dysfunction (CLAD). Using a two-colour fluorescent antigen microarray with 152 unique antigens, we previously observed that lung transplant recipients who developed CLAD exhibited persisting serum IgM reactivity to several tissue antigens. Among these we observed heightened reactivity to tropoelastin, a precursor of elastin, a major component of the lung extracellular matrix. Here we tested the hypothesis that higher reactivity to tropoelastin is associated with risks for de novo donor-specific antibodies (DSA) and CLAD <h3>Methods</h3> In a single center retrospective study, we measured IgG and IgM reactivities in pre-transplant and six months post-transplant serum samples of 96 flow crossmatch negative bilateral lung transplant recipients. Log2- transformed mean fluorescent intensities (MFIs) were determined for each antigen. Significance analysis of microarrays was used to analyze the results (false discovery rate of 0.05). CLAD was determined using published definitions. A univariate Cox proportional hazards model was performed to examine whether increases in anti-tropoelastin IgM reactivity post-transplant were associated with CLAD risk <h3>Results</h3> Samples were available for 93 patients: 36 (38.7%) patients developed CLAD and 57 (61.3%) patients remained CLAD-free by 5 years post-transplant. Patients with high pre-transplant IgM reactivity to tropoelastin had increased risk for the development of de novo donor specific antibodies (DSA) to HLA antigens post-transplant (Fig 1). A univariate Cox proportional hazards model showed that patients with an above-the-median pre-to-post-transplant increase in tropoelastin IgM reactivity had a shorter time to CLAD (Fig 2, HR 2.2, CI 1.1-4.5) <h3>Conclusion</h3> Serum IgM anti-tropoelastin antibodies present pre- and at 6 months post-transplant may enhance risks for DSA and CLAD. Further work will be required to validate this observation

Multiplexed autoantibody (AA) profiling of patients (pts) with metastatic urothelial carcinoma (mUC) receiving immune checkpoint inhibitors or platinum-based chemotherapy.

558 Background: The AA profile may be altered in malignancies and provide insights into tumor biology and the immune state. We hypothesized that the longitudinal AA profiling of mUC pts receiving an immune checkpoint inhibitor (ICI) may provide insights into the immune response, which may be associated with immune events and help discover new therapeutic targets. Methods: We utilized serum from mUC pts receiving an ICI or platinum-based chemotherapy (PBC) at the Dana-Farber Cancer Institute. Age and sex matched healthy controls were also studied. The SeroTag immuno-oncology discovery array (Oncimmune) was utilized, with quantification of the AA reactivity towards 1150 antigens. Bound autoantibodies were detected using an anti-IgG-specific detection antibody conjugated to the fluorescent reporter dye phycoerythrin. The AA reactivity was reported as the median fluorescence intensity (MFI) for each color and sample using a Luminex FlexMAP3D analyzer. A significance analysis of microarrays was performed to identify AAs with elevated levels in bladder cancer compared to matched healthy controls (HCs). AAs with &gt; 1.5 increase between pre- and post-treatment were reported. Scatter and box-whisker plots were reported for all pts and antigens, respectively. Results: Pre- (n = 66) and post treatment (n = 65) serum samples were available from mUC pts receiving pembrolizumab (n = 25), atezolizumab (n = 21), nivolumab (n = 5), avelumab (n = 1), durvalumab + tremelimumab (n = 1), nivolumab plus vaccine (n = 1), and 12 pts who received PBC (cisplatin n = 8, carboplatin n = 4). The median duration between the pre- and post-therapy samples was 6 months, median age was 67.7 years (range 40-91) with 51 men (77.3%). Overall, significant heterogeneity of AAs between pts was observed with 37 AAs showing higher reactivity in pre-treatment mUC pts vs. 47 HCs, notably anti-CTAG1 (NY-ESO-1), CTAG2 (NY-ESO-2), MAGE B-18, KRAS, GRB2, RARRES2, HSP72 and FGFR3 (all p &lt; 0.05). Pre- and post-therapy AA profiles were similar with unique changes seen in each patient. Notably, 3 pts receiving an ICI developed AAs to NY-ESO-1. Pts receiving PBC less frequently developed new AAs, although pts treated with cisplatin appeared to develop AAs more frequently compared to carboplatin-treated pts. Conclusions: This is the first report of a comprehensive AA profile using a novel platform in mUC pts. The study identified multiple elevated AAs in mUC pts vs. HCs, most notably NY-ESO-1, which also developed in some pts following ICIs. Pts treated with PBC did not develop new AAs frequently, although there appeared to be a difference between cisplatin and carboplatin-based chemotherapy. Further development of this platform is warranted to provide data that is orthogonal to genomic/transcriptomic profiling and shed insights on potential therapeutically actionable antigens.

Abstract P5-13-14: Antitumor activity of patritumab deruxtecan (HER3-DXd), a HER3-directed antibody drug conjugate (ADC) across a diverse panel of breast cancer (BC) patient-derived xenografts (PDXs)

Abstract Background: HER3 is overexpressed in 30-50% of breast cancers and has been associated with poor prognosis. Patritumab deruxtecan (HER3-DXd; U3-1402) is a HER3-directed ADC with a potent topoisomerase I (TOP1) inhibitor payload. A phase 1/2 study of HER3-DXd (NCT02980341) demonstrated promising antitumor activity in hormone receptor positive (HR+) metastatic BC patients with clinical activity observed across baseline levels of HER3 protein or mRNA expression. A window of opportunity clinical trial is currently ongoing to evaluate the biological activity of HER3-DXd in patients with treatment naïve BC according to HER3 mRNA/protein expression levels (NCT04610528). Here, we aimed to describe the activity of HER3-DXd in PDX models to identify robust biomarkers of response. Methods: The antitumor activity of HER3-DXd was assessed in 21 BC PDX models (14 HR+ and 7 triple negative). HER3-DXd sensitivity was established as a complete response that lasted longer than 120 days, following 4 weekly doses of 10 mg/kg. HER3-DXd antitumor activity was compared to the antitumor activity of irinotecan (50 mg/kg dosed once weekly), which was evaluated according to modified RECIST criteria in a subset of 14 BC PDX models. PDX models of acquired resistance to HER3-DXd were generated by repeated treatment cycles. Pharmacodynamic (PD) experiments were conducted by collecting tumor samples from PDXs after a single dose of HER3-DXd or irinotecan. Baseline HER3 expression was assessed by immunohistochemistry (IHC) and Western blot. mRNA expression of 72 genes including ERBB3 and genes from the PAM50 signature were measured using the nCounter platform. Two-class unpaired significance analysis of microarrays (SAM), using a false-discovery rate&amp;lt;5%, identified differential gene expression across response groups. Genetic alterations harbored by PDX models were determined using the MSK-IMPACTTM targeted exome panel. Quantification of proliferation (% of Ki67-positive cells) and of DNA damage during the S-phase of the cell cycle (γH2AX nuclear foci in geminin-positive cells) was evaluated in untreated/treated PD samples by IHC or immunofluorescence (IF), respectively. Western blot was used to assess HER3-pathway downmodulation and induction of apoptosis. Results: Eight out of 21 (38%) PDXs were highly sensitive to HER3-DXd, and in 5/14 (36%) models HER3-DXd showed a superior antitumor activity when compared to irinotecan. We observed an enrichment of basal-like models amongst the non-relapsed PDXs, compared to the relapsed ones (6/8 (75%) vs. 3/13 (20%), p=0.0195). Baseline levels of HER3/ERBB3 were not associated with treatment response and a model of acquired-resistance did not exhibit a reduction in baseline HER3 expression. Interestingly, relapsed models showed increased expression of genes related with chemotherapy-resistance (MDM2, NAT1, MAPT, GRB7, BCL-2). Mechanistically, treatment with HER3-DXd did not reduce the level of Ki67-proliferating cells but resulted in a significantly higher induction of S-phase DNA damage measured as γH2AX nuclear foci in non-relapsed models, compared to relapsed ones. This was accompanied by a reduction of HER3 protein levels and downmodulation of pERK1/2 T202/Y204, along with activation of PARP cleavage. Conclusions: HER3-DXd exerts a potent antitumor response in BC PDXs, independently of baseline HER3/ERBB3 levels, in line with the clinical data of NCT02980341. Basal-like tumors were more sensitive to HER3-DXd than luminal B models. Mechanistically, our data suggests that treatment with HER3-DXd results in parallel HER3/ERK signaling downmodulation and induction of S-phase DNA damage, resulting in tumor cell death. Citation Format: Andreu Òdena, Laia Monserrat, Fara Brasó-Maristany, Marta Guzmán, Judit Grueso, Olga Rodríguez, Maurizio Scaltriti, Sarat Chandarlapaty, Yang Qiu, Kumiko Koyama, Mafalda Oliveira, Aleix Prat, Violeta Serra. Antitumor activity of patritumab deruxtecan (HER3-DXd), a HER3-directed antibody drug conjugate (ADC) across a diverse panel of breast cancer (BC) patient-derived xenografts (PDXs) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-14.

Investigation of miRNA expression profiles using cohort samples reveals potential early detectability of colorectal cancers by serum miR-26a-5p before clinical diagnosis.

Previous studies have investigated the usefulness of microRNA (miRNA/miR) expression data for the early detection of colorectal cancer (CRC). However, limited data are available regarding miRNAs that detect CRC before clinical diagnoses. Accordingly, the present study investigated the early detectability of CRC by miRNAs using the preserved serum samples of the cohort participants affected with CRC within 2 years of study enrollment. First, the significant miRNAs were revealed using clinical CRC samples for a (seven early CRCs and seven controls) microarray analysis based on significance analysis of microarrays. Next, replicability was verified by reverse transcription-quantitative (RT-q)PCR (eight early CRCs and eight controls, together with 12 CRCs and 12 controls). Finally, early detectability was tested using the cohort samples of Japan Multi-Institutional Collaborative Cohort Study (17 CRCs and 17 controls) to reveal how a certain number of patients developed CRC within 2 years after participation. In the discovery phase, miRNA expression measurements were conducted using a 3D-Gene Human miRNA Oligo Chip for 2,555 miRNAs, and RT-qPCR analyses were performed to validate the replicability. In the first validation set with eight CRCs with early clinical stage and eight age- and gender-matched controls, miR-26a-5p and miR-223-3p demonstrated the highest diagnostic accuracy of area under the curve (AUC)=1.000 (sensitivity and specificity 100%). In an examination of the predictability of CRC incidence using pre-clinical cohort samples, miR-26a-5p demonstrated good predictability of advanced CRC incidence with an AUC of 0.840. Overall, the present study revealed serum miR-26a-5p as a potential early detection marker for CRC.

Regulatory effects of miR-28 on osteogenic differentiation of human bone marrow mesenchymal stem cells

ABSTRACT We aimed to assess the regulatory effects of miR-28 on the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMMSCs). HBMMSCs isolated, cultured and induced (at P3) to undergo osteogenic induction. The expressions of miRNAs were detected by gene microarray, and differentially expressed miRNAs in hBMMSCs compared with induced cells were obtained by significance analysis of microarrays. The microarray findings were confirmed by RT-PCR. TargetScan showed that signal transducer and activator of transcription 1 (STAT1) was the downstream target gene of miR-28. The relationship between miR-28 and STAT1 was validated using dual-luciferase reporter gene assay. HBMMSCs were transfected with miR-28 mimics and STAT1 siRNA, respectively. Samples were collected on day 10 after osteogenic differentiation, and the alkaline phosphatase (AKP) activity, Runt-related transcription factor 2 (RUNX2, a key regulator of osteogenic differentiation) and STAT1 expressions were determined using kits, PCR and Western blotting, respectively. Cell proliferation and migration were detected through CCK-8 and Transwell assays, respectively. During the osteogenic differentiation of hBMMSCs, the expression level of miR-28 increased. MiR-28 specifically bound the 3’-untranslated region (3’UTR) of STAT1 mRNA. It inhibited STAT1 expression in a targeted manner during osteogenic differentiation. Interference with STAT1 partially mimicked the regulatory effects of miR-28 overexpression on the osteogenic differentiation of hBMMSCs. Interference with STAT1 or overexpression of miR-28 did not affect proliferation or migration. MiR-28 has gradually increased expression during the osteogenic differentiation of hBMMSCs, which can directly bind STAT1 3’UTR and inhibit its expression, thereby up-regulating AKP and RUNX2, and promoting osteogenic differentiation.

Origin of indigo colorants revealed by ion mobility spectrometry coupled to mass spectrometry followed by supervised classification

Identification of colorants is a key theme in art history and restoration. Indigo, the famous blue dye and pigment, was historically produced from four biological sources cultivated in specific parts of the world: woad in Europe, true indigo in Indian region, anil in South and Central America, and Japanese indigo in Eastern Asia. Therefore, identification of the biological source of indigo could reveal the provenance of pigments and dyes used in historical artworks, assist evaluation of their age, and help to trace ancient trade routes. To date, none of the previous reports achieved confident identification of indigo sources. This is the first systematic comprehensive untargeted workflow for discrimination of indigo colorants from different biological sources. Besides the new analytical procedure based on flow injection analysis and high-resolution mass spectrometry coupled to ion mobility spectrometry, the original multi-method screening scheme for feature selection, classification model training, and validation is presented. Reference indigo pigments were analyzed neat or embedded into appropriate binders to simulate oil painting or egg tempera mural. Combinations of eight feature selection methods (information gain, information gain ratio, Gini decrease, ANOVA, Chi squared, ReliefF, fast correlation based filter, and significance analysis of microarrays) and five classification algorithms (k-nearest neighbors, support-vector machines, random forest, neural network, and naïve Bayes) were evaluated via double (nested) cross-validation. The best performance was achieved with random forest across all feature selection methods (average classification accuracy 94%, negative ionization mode data). Tentative substance classes were proposed for the most important features based on their mass fragmentation patterns. A methylated flavonoid (diosmetin) and an indole alkaloid (2,2′-biindolyl) were identified in woad and true indigo pigments, respectively, and confirmed by chromatography. Plant sources of colorants were determined in historical samples of oil paintings and textiles.

Common Gene Expression Signature of B-Cells of Waldenström Macroglobulinemia (WM) and IgM Monoclonal Gammopathies of Undetermined Significance (IgM MGUS) Compared to Healthy Subjects

We performed a comparative gene expression profiling (GEP) study on B-cells and plasma cells of Waldenström Macroglobulinemia (WM), IgM monoclonal gammopathies of undetermined significance (IgMMGUS), and normal individuals (CTRLs) to identify GEP changes as reliable predictors of progression of IgMMGUS to WM.We analyzed bone marrow B-cells and plasma cells from 36 WM patients, 13 IgMMGUS subjects, and 7 CTRLs by Affymetrix microarray, respectively (Table 1). GEP experiments were performed on the CD19+ and CD138+ cells using GeneChip-HGU133 Plus 2.0. Data were preprocessed and normalized by Robust Multi-Array Average and ComBat. Selection of the different expressed genes was performed separately for CD19+ and CD138+ cells, using Significance Analysis of Microarrays (SAM) on the 3 groups and a false discovery rate threshold of 5%, followed, for significance comparisons, by a pair-wise SAM test corrected for multiple testing.We focused on the comparison of the CD19+ cells of WM vs. IgMMGUS vs. CTRLs which highlighted 2038 unique genes whereas the same comparison of the CD138+ cells determined 29 unique genes (Trojani et.al. Cancers 2021). Among the 2038 DEGs, 115 genes were grouped in KEGG pathways involved in Wnt-signaling, BCR-signaling, calcium signaling, hematopoietic cell antigens, cell adhesion, adherens junctions, coagulation cascade, platelet activation, cytokine receptor, and signaling pathways responsible for cell cycle, apoptosis, and survival.Interestingly, most of the 115 DEGs in B-cells were different expressed in WM vs. IgMMGUS and CTRLs. Only 9/115 DEGs were significantly different expressed in WM vs. CTRLs and in IgMMGUS vs. CTRLs, but no significant expression changes were noted between WM and IgMMGUS (Table 2).To further inspect the similarities and the differences among WM and IgMMGUS, we computed the Euclidean pair-wise distance between subjects and, using this distance as weight, constructed a minimum spanning tree (MST) (Figure 1).Considerably, four probesets identified ADRB2 (transmembrane Beta adrenergic receptor) which was up regulated in WM and IgMMGUS compared to CTRLs. The over expression of ADRB2 was also demonstrated in Mantle Cell Lymphoma cell lines and in Diffuse large B-cell lymphoma (DLBCL) lymphocytes compared to normal B-cells (doi10.1016/j.cellsig.2017.08.002), and in most malignancies (doi10.1007/s11033-021-06250-y) .As far as we know, ADAM23 (ADAM Metallopeptidase Domain23) has not been found in WM, whereas we suggest its possible role in WM patients with Sjogren's syndrome (SS). ADAM23 plays a role in the peripheral neuropathy by controlling the activity of potassium channels in SS (doi10.1007/s10067-016-3499-z). Some authors found that sensory/motor neuropathies were associated with MGUS patients (doi10.1017/s0317167100011483). We strongly believe that the down regulation of ADAM23 in WM and IgMMGUS has a good chance to be associated with clinical neuropathy in WM and IgMMGUS.RASGRP3 (RAS Guanyl Releasing Protein3) and PIK3AP1 (Phosphoinositide 3 Kinase Adaptor Protein1) play crucial roles in BCR signaling pathway: PIK3AP1 activates the PI3K-Akt signaling while RASGRP3 stimulates MAPK signaling pathway.The deregulation of LEF1 (Lymphoid Enhancer Binding Factor1) and genes of Wnt-pathway were previously demonstrated in B-cell disorders and multiple myeloma (doi10.1007/s00277-017-3207-3, doi10.1016/j.pathol.2019.09.009). According to these studies, we showed the under expression of LEF1 in WM and IgMMGUS compared to CTRLs.We identified the down regulation of EZH2 (Enhancer Of Zeste2 Polycomb Repressive Complex2Subunit) in WM and IgMMGUS compared to CTRLs. EZH2 is involved in Follicular lymphoma and DLBCL (doi10.1080/2162402X.2017.1321184).CDHR3 (Cadherin Related Family Member3), CHEK1 (Checkpoint Kinase1), and HIST1H1B (Histone-H1.5) were over expressed in CTRLs compared to IgMMGUS and WM.In conclusion, the common gene-set in WM and IgMMGUS could suggest two-hit hypothesis. First, the gene-set could play a role in the risk of progression of IgMMGUS to WM. Until now, all the IgMMGUS subjects have not been transformed in WM or other NHL, but they have been monitored every 6 months, and their possible transformation to lymphoma could highlight new insights. The second hypothesis suggests their involvement in the biological processes of leukemogenesis in WM and IgMMGUS which will be further investigated. [Display omitted] DisclosuresTedeschi: AbbVie: Honoraria, Speakers Bureau; AstraZeneca: Honoraria, Speakers Bureau; Beigene: Honoraria, Speakers Bureau; Janssen: Honoraria, Speakers Bureau.

Molecular characterization of triple negative breast cancer formaldehyde-fixed paraffin-embedded samples by data-independent acquisition proteomics.

Triple negative breast cancer accounts for 15%-20% of all breast carcinomas and is clinically characterized by an aggressive phenotype and poor prognosis. Triple negative tumors do not benefit from targeted therapies, so further characterization is needed to define subgroups with potential therapeutic value. In this work, the proteomes of 125 formalin-fixed paraffin-embedded samples from patients diagnosed with non-metastatic triple negative breast cancer were analyzed using data-independent acquisition + in a LTQ-Orbitrap Fusion Lumos mass spectrometer coupled to an EASY-nLC 1000. 1206 proteins were identified in at least 66% of the samples. Hierarchical clustering, probabilistic graphical models and Significance Analysis of Microarrays were combined to characterize proteomics-based molecular groups. Two molecular groups were defined with differences in biological processes such as glycolysis, translation and immune response. These two molecular groups showed also several differentially expressed proteins. This clinically homogenous dataset may serve to design new therapeutic strategies in the future.

Serum miRNA Profiling for Early PDAC Diagnosis and Prognosis: A Retrospective Study.

Background: Tumor stage predicts pancreatic cancer (PDAC) prognosis, but prolonged and short survivals have been described in patients with early-stage tumors. Circulating microRNA (miRNA) are an emerging class of suitable biomarkers for PDAC prognosis. Our aim was to identify whether serum miRNA signatures predict survival of early-stage PDAC. Methods: Serum RNA from archival 15 stage I-III PDAC patients and 4 controls was used for miRNAs expression profile (Agilent microarrays). PDAC patients with comparable age, gender, diabetes, jaundice and surgery were classified according to survival: less than 14 months (7/15 pts, group A) and more than 22 months (8/15 pts, group B). Bioinformatic data analysis was performed by two-class Significance Analysis of Microarray (SAM) algorithm. Binary logistic regression analyses considering PDAC diagnosis and outcome as dependent variables, and ROC analyses were also performed. Results: 2549 human miRNAs were screened out. At SAM, 76 differentially expressed miRNAs were found among controls and PDAC (FDR = 0.4%), the large majority (50/76, 66%) of them being downregulated in PDAC with respect to controls. Six miRNAs were independently correlated with early PDAC, and among these, hsa-miR-6821-5p was associated with the best ROC curve area in distinguishing controls from early PDAC. Among the 71 miRNAs differentially expressed between groups A and B, the most significant were hsa-miR-3135b expressed in group A only, hsa-miR-6126 and hsa-miR-486-5p expressed in group B only. Eight miRNAs were correlated with the presence of lymph-node metastases; among these, hsa-miR-4669 is of potential interest. hsa-miR-4516, increased in PDAC and found as an independent predictor of survival, has among its putative targets a series of gens involved in key pathways of cancer progression and dissemination, such as Wnt and p53 signalling pathways. Conclusions: A series of serum miRNAs was identified as potentially useful for the early diagnosis of PDAC, and for establishing a prognosis.

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.