Characteristic Gene Signature Research Articles

Seventeen-Gene Signature Identifies AML Cells in Single Cell RNA Sequencing Data and Resolves Treatment Resistant Subtypes

Background: Pediatric acute myeloid leukemia (AML) is a highly heterogeneous malignancy with disparate risk profiles between subtypes. While the advent of single-cell technologies enabled granular evaluation of disease biology, differentiation of malignant cells from healthy analogs remains challenging. Reliable identification of malignant cells has the potential to enhance transcriptomic characterization of high-risk AML subtypes. Methods: We applied Support Vector Machine (SVM) learning to four single-cell RNA sequencing (scRNAseq) datasets (101 bone marrow biopsies: 75 diagnoses, 15 post-treatment, 11 healthy controls) containing 204,836 cells to develop a geneset for discrimination of malignant and non-malignant cells. AML cells were identified based on canonical gene expression, patient occupancy scoring, clustering relative to healthy bone marrow, and inferred alterations (inferCNV) for known copy number variants. AML subtype clusters that persisted in end-of-induction (EOI) and relapse were correlated with clinical outcomes and underwent differential expression to identify high-risk transcriptomic profiles. High-risk subtypes were validated by the application of characteristic gene signatures to large bulk RNA sequencing datasets from the Therapeutically Applicable Research to Generate Effective Treatments initiative and National Center for Biotechnology Information Gene Expression Omnibus. Results: SVM classification using a 17-geneset discriminated malignant from non-malignant cells with areas under the curve from 0.84-0.95 (ARMH1, CLEC11A, NREP, AZU1, PRAME, IFITM2, CFD, DUSP6, SCN3A, CD163, SOX4, IFI30, HOXA9, CD44, SRGN, UBE2C, CXCL8). Malignant cells grouped into 1-5 clusters of monocyte-like, hematopoietic-stem-cell-like (HSC), granulocyte-monocyte-progenitor-like (GMP) AML cells. One HSC-like and one monocyte-like AML cluster persisted through treatment with both a > 1.5-fold proportional enrichment at EOI or relapse and at least a 50% clinical relapse rate. Differential expression (log 2FC>1, adjusted p<0.05) yielded characteristic gene-sets (HSC: 20 genes, Mono: 56 genes). The high-risk HSC subtype showed increased expression of genes associated with chemoresistance (CD69, Log 2FC: 1.46, adjusted p = 3.48x10 -50), angiogenesis (MDK: Log 2FC = 1.04, adjusted p = 9.25 x 10 -50), and poor survival (BCL3: Log 2FC = 1.18, adjusted p = 1.00 x 10 -47). Patients with similar transcriptomes in bulk RNAseq had worse overall survival with hazard ratios from 1.31-3.35 across datasets (Cox PH p-values 4.38x10 -2 to 7.20x10 -3). Conclusions: These findings illustrate that malignant AML cells can be differentiated from healthy analogs solely based on gene expression. Application of our 17-geneset revealed two high-risk transcriptomic profiles, with the HSC subtype having particularly poor outcomes. From this foundation, future work will identify malignant transcriptomic changes amenable to established chemotherapeutics.

High and Low Levels of ABCB1 Expression Are Associated with Two Distinct Gene Signatures in Lung Tissue of Pulmonary TB Patients with High Inflammation Activity.

P-glycoprotein (encoded by the ABCB1 gene) has a dual role in regulating inflammation and reducing chemotherapy efficacy in various diseases, but there are few studies focused on pulmonary TB patients. In this study, our objective was to identify a list of genes that correlate with high and low levels of ABCB1 gene expression in the lungs of pulmonary TB patients with different activity of chronic granulomatous inflammation. We compared gene expression in two groups of samples (with moderate and high activity of tuberculomas) to identify their characteristic gene signatures. Gene expression levels were determined using quantitative PCR in samples of perifocal area of granulomas, which were obtained from 65 patients after surgical intervention. Subsequently, two distinct gene signatures associated with high inflammation activity were identified. The first signature demonstrated increased expression of HIF1a, TGM2, IL6, SOCS3, and STAT3, which correlated with high ABCB1 expression. The second signature was characterized by high expression of TNFa and CD163 and low expression of ABCB1. These results provide insight into various inflammatory mechanisms and association with P-gp gene expression in lung tissue of pulmonary TB patients and will be useful in the development of a host-directed therapy approach to improving the effectiveness of anti-TB treatment.

CD39 and LDHA affects the prognostic role of NLR in metastatic melanoma patients treated with immunotherapy

BackgroundIdentifying response markers is highly needed to guide the treatment strategy in patients with metastatic melanoma.MethodsA retrospective study was carried out in patients with unresectable/metastatic melanoma (stage IIIb–IV), treated with anti-PD-1 in the first line setting, to better explore the role and the timing of neutrophil/lymphocyte ratio (NLR) as potential biomarker of response. The relationship of NLR with inflammation-immune mediators and the underlying negative effect of raising NLR during immunotherapy, have been investigated with transcriptomic gene analysis.ResultsThe results confirmed previous findings that a high baseline NLR is associated with a poorer prognosis and with higher serum level of lactate dehydrogenase (LDH), regardless of the presence of brain metastases. The transcriptomic analysis showed that high baseline NLR is associated with a characteristic gene signature CCNA1, LDHA and IL18R1, which correlates with inflammation and tumorigenesis. Conversely, low baseline NLR is associated with the signature CD3, SH2D1A, ZAP70 and CD45RA, linked to the immune-activation. The genes positively associated with NLR (CD39 (ENTPD1), PTEN, MYD88, MMP9 and LDH) are involved in processes of immunosuppression, inflammation and tumor-promoting activity. Increased expression of CD39 correlated with TGFβ2, a marker of the N2 neutrophils with immunosuppressive activity.ConclusionsThese results suggest that increasing NLR is associated with an increased neutrophil population, with polarization to the N2 phenotype, and this process may be the basis for the negatively prognostic role of NLR.

Construction of a Matrix Cancer-Associated Fibroblast Signature Gene-Based Risk Prognostic Signature for Directing Immunotherapy in Patients with Breast Cancer Using Single-Cell Analysis and Machine Learning.

Cancer-associated fibroblasts (CAFs) are heterogeneous constituents of the tumor microenvironment involved in the tumorigenesis, progression, and therapeutic responses of tumors. This study identified four distinct CAF subtypes of breast cancer (BRCA) using single-cell RNA sequencing (RNA-seq) data. Of these, matrix CAFs (mCAFs) were significantly associated with tumor matrix remodeling and strongly correlated with the transforming growth factor (TGF)-β signaling pathway. Consensus clustering of The Cancer Genome Atlas (TCGA) BRCA dataset using mCAF single-cell characteristic gene signatures segregated samples into high-fibrotic and low-fibrotic groups. Patients in the high-fibrotic group exhibited a significantly poor prognosis. A weighted gene co-expression network analysis and univariate Cox analysis of bulk RNA-seq data revealed 17 differential genes with prognostic values. The mCAF risk prognosis signature (mRPS) was developed using 10 machine learning algorithms. The clinical outcome predictive accuracy of the mRPS was higher than that of the conventional TNM staging system. mRPS was correlated with the infiltration level of anti-tumor effector immune cells. Based on consensus prognostic genes, BRCA samples were classified into the following two subtypes using six machine learning algorithms (accuracy > 90%): interferon (IFN)-γ-dominant (immune C2) and TGF-β-dominant (immune C6) subtypes. Patients with mRPS downregulation were associated with improved prognosis, suggesting that they can potentially benefit from immunotherapy. Thus, the mRPS model can stably predict BRCA prognosis, reflect the local immune status of the tumor, and aid clinical decisions on tumor immunotherapy.

MP20-01 PROTEIN KINASE D1 (PrKD) PROTECTS AGAINST DNA DAMAGE BY INCREASING Α-CATENIN LEVELS THROUGH PUTATIVE SERINE681 AND 685 PHOSPHORYLATION OF CYCLIN DEPENDENT KINASE 12 (CDK12) IN PROSTATE CANCER (PC)

You have accessJournal of UrologyCME1 Apr 2023MP20-01 PROTEIN KINASE D1 (PrKD) PROTECTS AGAINST DNA DAMAGE BY INCREASING Α-CATENIN LEVELS THROUGH PUTATIVE SERINE681 AND 685 PHOSPHORYLATION OF CYCLIN DEPENDENT KINASE 12 (CDK12) IN PROSTATE CANCER (PC) Sanjeev Shukla, Teruko Osumi, Mohammed Al-Toubat, Carlos Riveros, Allison H. Feibus, Ahmed El-shafie, Adam M. Kase, John A. Copland, Joachim L. Petit, Nathalie Meurice, Justyna Gleba, Jonathan Chardon-Robles, and K. C. Balaji Sanjeev ShuklaSanjeev Shukla More articles by this author , Teruko OsumiTeruko Osumi More articles by this author , Mohammed Al-ToubatMohammed Al-Toubat More articles by this author , Carlos RiverosCarlos Riveros More articles by this author , Allison H. FeibusAllison H. Feibus More articles by this author , Ahmed El-shafieAhmed El-shafie More articles by this author , Adam M. KaseAdam M. Kase More articles by this author , John A. CoplandJohn A. Copland More articles by this author , Joachim L. PetitJoachim L. Petit More articles by this author , Nathalie MeuriceNathalie Meurice More articles by this author , Justyna GlebaJustyna Gleba More articles by this author , Jonathan Chardon-RoblesJonathan Chardon-Robles More articles by this author , and K. C. BalajiK. C. Balaji More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003245.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: PrKD is protective of DNA damage in PC and loss of PrKD leading to increased genomic instability is a novel potential mechanism. CDK12 mutated PC is now recognized as a distinct aggressive variant associated with genomic instability. We explored whether PrKD influences DNA damage through CDK12. METHODS: The DNA damage and protein levels studied by COMET assay and western blot respectively and validated the findings in human PC samples by querying cBioPortal gene expression. RESULTS: PrKD abrogated etoposide induced DNA damage in castration insensitive C4-2 PC cells. PrKD is a membrane bound protein that phosphorylates proteins in cell adherens junction including β-catenin. As PrKD is the only known kinase to phosphorylate T120 in β-catenin, mutation of T120 leads to nuclear translocation of transcriptionally active β-catenin playing a role in DNA damage, uncouples the binding of β- to α-catenin, which translocates to the nucleus and influences β-catenin. PrKD increased the expression of α-catenin, which was attenuated by CDK12 wild type and double non-phosphorylated mutants of PrKD putative phosphorylation sites; serine (s) 681 and 685 but not as much by s681 mutant. PrKD suppressed the substrate phosphorylation of γH2AX s139 by CDK12 s681 and s681/s685 non-phosphorylated mutant compared to wild type, which links PrKD substrate phosphorylation of CDK12 to DNA damage. Query of cBioPortal for concurrent PrKD1 and CDK12 mutations in PC with characteristic gene signature of DNA damage repair (DDR) revealed the highest concordance of PrKD1 and CDK12 mutations in patients with BRAC2 mutations and the DDR genes expression were not significantly different between patients with PrKD1 and CDK12 mutated patient except BRAC1 expression (rarely mutated in PC) suggesting that PrKD1 and CDK12 could be involved in the same signaling pathway (Figure 1). CONCLUSIONS: The study provides a validated mechanistic basis for the novel protective role of PrKD in DNA damage, which has potential implications in PC therapeutics linked to DNA damage. Source of Funding: none © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e275 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Sanjeev Shukla More articles by this author Teruko Osumi More articles by this author Mohammed Al-Toubat More articles by this author Carlos Riveros More articles by this author Allison H. Feibus More articles by this author Ahmed El-shafie More articles by this author Adam M. Kase More articles by this author John A. Copland More articles by this author Joachim L. Petit More articles by this author Nathalie Meurice More articles by this author Justyna Gleba More articles by this author Jonathan Chardon-Robles More articles by this author K. C. Balaji More articles by this author Expand All Advertisement PDF downloadLoading ...

Developmental Heterogeneity of Microglia and Brain Myeloid Cells Revealed by Deep Single-Cell RNA Sequencing

Microglia are increasingly recognized for their major contributions during brain development and neurodegenerative disease. It is currently unknown whether these functions are carried out by subsets of microglia during different stages of developmentand adulthood or within specific brain regions. Here, we performed deep single-cell RNA sequencing (scRNA-seq) of microglia and related myeloid cells sorted from various regions of embryonic, early postnatal, and adult mouse brains. We found that the majority of adult microglia expressing homeostatic genes are remarkably similar in transcriptomes, regardless of brain region. By contrast, early postnatal microglia are more heterogeneous. We discovered a proliferative-region-associated microglia (PAM) subset, mainly found in developing white matter, that shares a characteristic gene signature with degenerative disease-associated microglia (DAM). Such PAM have amoeboid morphology, are metabolically active, and phagocytose newly formed oligodendrocytes. This scRNA-seq atlas will be a valuable resource for dissecting innate immune functions in health and disease.

Deciphering hepatocellular responses to metabolic and oncogenic stress.

Each cell type responds uniquely to stress and fractionally contributes to global and tissue-specific stress responses. Hepatocytes, liver macrophages (MΦ), and sinusoidal endothelial cells (SEC) play functionally important and interdependent roles in adaptive processes such as obesity and tumor growth. Although these cell types demonstrate significant phenotypic and functional heterogeneity, their distinctions enabling disease-specific responses remain understudied. We developed a strategy for the simultaneous isolation and quantification of these liver cell types based on antigenic cell surface marker expression. To demonstrate the utility and applicability of this technique, we quantified liver cell-specific responses to high-fat diet (HFD) or diethylnitrosamine (DEN), a liver-specific carcinogen, and found that while there was only a marginal increase in hepatocyte number, MΦ and SEC populations were quantitatively increased. Global gene expression profiling of hepatocytes, MΦ and SEC identified characteristic gene signatures that define each cell type in their distinct physiological or pathological states. Integration of hepatic gene signatures with available human obesity and liver cancer microarray data provides further insight into the cell-specific responses to metabolic or oncogenic stress. Our data reveal unique gene expression patterns that serve as molecular "fingerprints" for the cell-centric responses to pathologic stimuli in the distinct microenvironment of the liver. The technical advance highlighted in this study provides an essential resource for assessing hepatic cell-specific contributions to metabolic and oncogenic stress, information that could unveil previously unappreciated molecular mechanisms for the cellular crosstalk that underlies the continuum from metabolic disruption to obesity and ultimately hepatic cancer.

Clear cell sarcoma of the kidney demonstrates an embryonic signature indicative of a primitive nephrogenic origin

Clear cell sarcoma of the kidney (CCSK) is a tumor affecting children with a median age of 3 years at diagnosis. The cell of origin of CCSK is unknown and data on the molecular changes giving rise to CCSK is scarce. This has hindered the identification of positive diagnostic markers and development of molecularly targeted treatment protocols for CCSK. We have characterized a panel of CCSK to gain information regarding its molecular profile and possible origin. High-resolution genomic analysis with single nucleotide polymorphism array of 37 tumors did not reveal any clues to the mechanisms behind tumor development as remarkably few genetic imbalances were found. Gene expression analysis revealed a highly characteristic gene signature, enriched for pathways involved in embryonic development, including kidney formation. The presence of markers for two different developmental lineages in the embryonic kidney was therefore investigated in the tumor cells. FOXD1 which identifies cells giving rise to stromal elements, and CITED1, a marker for cells primed for nephrogenic epithelial differentiation, were both highly expressed in CCSK. In addition, the early embryonic marker OSR1 was expressed at higher levels in CCSK than in Wilms tumor, normal fetal kidney or adult kidney. As this marker discriminates the intermediate mesoderm from other mesodermal structures, our study could suggest that CCSK arises from a mesodermal cell type that retains the capacity to initiate differentiation towards both nephrons and stroma, but remains locked in a primitive state.

Molecular signatures associated with HCV-induced hepatocellular carcinoma and liver metastasis.

Hepatocellular carcinomas (HCCs) are a heterogeneous group of tumors that differ in risk factors and genetic alterations. In Italy, particularly Southern Italy, chronic hepatitis C virus (HCV) infection represents the main cause of HCC. Using high-density oligoarrays, we identified consistent differences in gene-expression between HCC and normal liver tissue. Expression patterns in HCC were also readily distinguishable from those associated with liver metastases. To characterize molecular events relevant to hepatocarcinogenesis and identify biomarkers for early HCC detection, gene expression profiling of 71 liver biopsies from HCV-related primary HCC and corresponding HCV-positive non-HCC hepatic tissue, as well as gastrointestinal liver metastases paired with the apparently normal peri-tumoral liver tissue, were compared to 6 liver biopsies from healthy individuals. Characteristic gene signatures were identified when normal tissue was compared with HCV-related primary HCC, corresponding HCV-positive non-HCC as well as gastrointestinal liver metastases. Pathway analysis classified the cellular and biological functions of the genes differentially expressed as related to regulation of gene expression and post-translational modification in HCV-related primary HCC; cellular Growth and Proliferation, and Cell-To-Cell Signaling and Interaction in HCV-related non HCC samples; Cellular Growth and Proliferation and Cell Cycle in metastasis. Also characteristic gene signatures were identified of HCV-HCC progression for early HCC diagnosis.ConclusionsA diagnostic molecular signature complementing conventional pathologic assessment was identified.

Transcriptomic responses generated by hepatocarcinogens in a battery of liver-based in vitro models

As the conventional approach to assess the potential of a chemical to cause cancer in humans still includes the 2-year rodent carcinogenicity bioassay, development of alternative methodologies is needed. In the present study, the transcriptomics responses following exposure to genotoxic (GTX) and non-genotoxic (NGTX) hepatocarcinogens and non-carcinogens (NC) in five liver-based in vitro models, namely conventional and epigenetically stabilized cultures of primary rat hepatocytes, the human hepatoma-derived cell lines HepaRG and HepG2 and human embryonic stem cell-derived hepatocyte-like cells, are examined. For full characterization of the systems, several bioinformatics approaches are employed including gene-based, ConsensusPathDB-based and classification analysis. They provide convincingly similar outcomes, namely that upon exposure to carcinogens, the HepaRG generates a gene classifier (a gene classifier is defined as a selected set of characteristic gene signatures capable of distinguishing GTX, NGTX carcinogens and NC) able to discriminate the GTX carcinogens from the NGTX carcinogens and NC. The other in vitro models also yield cancer-relevant characteristic gene groups for the GTX exposure, but some genes are also deregulated by the NGTX carcinogens and NC. Irrespective of the tested in vitro model, the most uniformly expressed pathways following GTX exposure are the p53 and those that are subsequently induced. The NGTX carcinogens triggered no characteristic cancer-relevant gene profiles in all liver-based in vitro systems. In conclusion, liver-based in vitro models coupled with transcriptomics techniques, especially in the case when the HepaRG cell line is used, represent valuable tools for obtaining insight into the mechanism of action and identification of GTX carcinogens.

Genes Involved in Viral Carcinogenesis and Tumor Initiation in Hepatitis C Virus-Induced Hepatocellular Carcinoma

The role of chronic hepatitis C virus (HCV) in the pathogenesis of HCV-associated hepatocellular carcinoma (HCC) remains controversial. To understand the transition from benign to malignant, we studied the gene expression patterns in liver tissues at different stages, including normal, cirrhosis, and different HCC stages. We studied 108 liver tissue samples obtained from 88 distinct patients (41 HCV-cirrhotic tissues, 17 HCV-cirrhotic tissues from patients with HCC, and 47 HCV-HCC tissues). Differentially expressed genes (DEG) were studied by use of high-density oligonucleotide arrays. Among probe sets identified as differentially expressed via the F test, all pairwise comparisons were performed. Cirrhotic tissues with and without concomitant HCC were further evaluated, and a classifier was used to predict whether the tissue type was associated with HCC. Differential expression profiles were analyzed using Interaction Networks and Functional Analysis. Characteristic gene signatures were identified when normal tissue was compared with cirrhosis, cirrhosis with early HCC, and normal with HCC. Pathway analysis classified the cellular and biological functions of the DEG as related to cellular growth and proliferation, cell death and inflammatory disease in cirrhosis; cell death, cell cycle, DNA replication, and immune response in early HCCs; and cell death, cell growth and proliferation, cell cycle, and DNA repair in advanced HCCs. Characteristic gene signatures were identified at different stages of HCV-HCC progression. A set of genes were identified to predict whether the cirrhotic tissue was associated with HCC.

Wnt/β-catenin signaling controls development of the blood–brain barrier

The blood–brain barrier (BBB) is confined to the endothelium of brain capillaries and is indispensable for fluid homeostasis and neuronal function. In this study, we show that endothelial Wnt/β-catenin (β-cat) signaling regulates induction and maintenance of BBB characteristics during embryonic and postnatal development. Endothelial specific stabilization of β-cat in vivo enhances barrier maturation, whereas inactivation of β-cat causes significant down-regulation of claudin3 (Cldn3), up-regulation of plamalemma vesicle-associated protein, and BBB breakdown. Stabilization of β-cat in primary brain endothelial cells (ECs) in vitro by N-terminal truncation or Wnt3a treatment increases Cldn3 expression, BBB-type tight junction formation, and a BBB characteristic gene signature. Loss of β-cat or inhibition of its signaling abrogates this effect. Furthermore, stabilization of β-cat also increased Cldn3 and barrier properties in nonbrain-derived ECs. These findings may open new therapeutic avenues to modulate endothelial barrier function and to limit the devastating effects of BBB breakdown.

Spanish customized adjuvant trial (SCAT) based on BRCA1 mRNA levels

7533 Background: Patients (p) with low BRCA1 mRNA levels attained longer survival when treated with cisplatin-based chemotherapy (Taron et al. 2004), while p with high BRCA1 levels attained longer survival when treated with taxanes-based therapy (Quinn et al. 2007). Early NSCLC p whose tumors had high BRCA1 levels had significantly worse survival (Rosell et al. 2007). Upregulated BRCA1 is part of a characteristic gene signature in aggressive lung cancers (Deeb et al. 2007). Methods: Adjuvant chemotherapy was customized based on BRCA1 mRNA levels in 84/100 completely resected N1 and N2 NSCLC p. 11 p with high BRCA1 levels received docetaxel (doc); 29 p with intermediate BRCA1 levels received doc/cisplatin (cis); 44 p with low BRCA1 levels received cis/gemcitabine (gem). Results: Median survival has not been reached in p with high or intermediate BRCA1 levels, while it is 25.6 months (m) in p with low levels (P=0.04). In a multivariate analysis for survival in all 84 p, the hazard ratios (HR) were 5.23 for p with high BRCA1 levels (P=0.07), 3.57 for p with tumor size > 4 cm (P=0.07). In the multivariate analysis of 42 N2 p, the HRs were 0.13 for 18 p receiving postoperative radiotherapy (P=0.04) and 22 for 15 p with intermediate or high BRCA1 levels (P=0.01). Conclusions: Interim analyses show that single-agent doc has no detrimental effect on survival in comparison with doc/cis. In addition, high BRCA1 mRNA expression could be a poor prognostic marker. The Spanish Lung Cancer Group has opened a phase III study of customized adjuvant chemotherapy based on BRCA1 mRNA levels. N2 p HR (95% CI) p Post-operative radiotherapy Yes 18 0.12 (0.02–0.89) 0.04 No 24 1 BRCA1 mRNA levels Low 24 1 Intermediate/High 15 22.43 (2.02–248.62) 0.01 Tumor size ≤ 4 24 1 > 4 18 1.63 (0.34- 7.91) 0.54 No significant financial relationships to disclose.