| 1. |
- Branca, Rui M. M., et al.
(author)
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HiRIEF LC-MSMS enables deep proteome coverage and unbiased proteogenomics
- 2014
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In: Nature Methods. - 1548-7091 .- 1548-7105. ; 11:1, s. 59-
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Journal article (peer-reviewed)abstract
- We present a liquid chromatography-mass spectrometry (LC-MSMS)-based method permitting unbiased (gene prediction-independent) genome-wide discovery of protein-coding loci in higher eukaryotes. Using high-resolution isoelectric focusing (HiRIEF) at the peptide level in the 3.7-5.0 pH range and accurate peptide isoelectric point (pI) prediction, we probed the six-reading-frame translation of the human and mouse genomes and identified 98 and 52 previously undiscovered protein-coding loci, respectively. The method also enabled deep proteome coverage, identifying 13,078 human and 10,637 mouse proteins.
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| 2. |
- Eriksson, Hanna, et al.
(author)
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Quantitative membrane proteomics applying narrow range peptide isoelectric focusing for studies of small cell lung cancer resistance mechanisms
- 2008
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In: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861. ; 28:5C, s. 3275-3276
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Journal article (peer-reviewed)abstract
- Drug resistance is often associated with upregulation of membrane-associated drug-efflux systems, and thus global membrane proteomics methods are valuable tools in the search for novel components of drug resistance phenotypes. Herein we have compared the microsomal proteome from the lung cancer cell line H69 and its isogenic Doxorubicin-resistant subcell line H69AR. The method used includes microsome preparation, iTRAQ labeling followed by narrow range peptide IEF in an immobilized pH-gradient (IPG-IEF) and LC-MS/MS analysis. We demonstrate that the microsomal preparation and iTRAQ labeling is reproducible regarding protein content and composition. The rationale using narrow range peptide IPG-IEF separation is demonstrated by its ability to: (i) lowering the complexity of the sample by two-thirds while keeping high proteome coverage (96%), (ii) providing high separation efficiency, and (iii) allowing for peptide validation and possibly identifications of post-transcriptional modifications. After analyzing one-fifth of the IEF fractions (effective pH range of 4.0-4.5), a total of 3704 proteins were identified, among which 527 were predicted to be membrane proteins. One of the proteins found to be differentially expressed was Serca 2, a calcium pump located in the ER membrane that potentially could result in changes of apoptotic response toward Doxorubicin.
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| 3. |
- Fotouhi, Omid, et al.
(author)
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Proteomics identifies neddylation as a potential therapy target in small intestinal neuroendocrine tumors
- 2019
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In: Oncogene. - : NATURE PUBLISHING GROUP. - 0950-9232 .- 1476-5594. ; 38:43, s. 6881-6897
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Journal article (peer-reviewed)abstract
- Patients with small intestinal neuroendocrine tumors (SI-NETs) frequently develop spread disease; however, the underlying molecular mechanisms of disease progression are not known and effective preventive treatment strategies are lacking. Here, protein expression profiling was performed by HiRIEF-LC-MS in 14 primary SI-NETs from patients with and without liver metastases detected at the time of surgery and initial treatment. Among differentially expressed proteins, overexpression of the ubiquitin-like protein NEDD8 was identified in samples from patients with liver metastasis. Further, NEDD8 correlation analysis indicated co-expression with RBX1, a key component in cullin-RING ubiquitin ligases (CRLs). In vitro inhibition of neddylation with the therapeutic agent pevonedistat (MLN4924) resulted in a dramatic decrease of proliferation in SI-NET cell lines. Subsequent mass spectrometry-based proteomics analysis of pevonedistat effects and effects of the proteasome inhibitor bortezomib revealed stabilization of multiple targets of CRLs including p27, an established tumor suppressor in SI-NET. Silencing of NEDD8 and RBX1 using siRNA resulted in a stabilization of p27, suggesting that the cellular levels of NEDD8 and RBX1 affect CRL activity. Inhibition of CRL activity, by either NEDD8/RBX1 silencing or pevonedistat treatment of cells resulted in induction of apoptosis that could be partially rescued by siRNA-based silencing of p27. Differential expression of both p27 and NEDD8 was confirmed in a second cohort of SI-NET using immunohistochemistry. Collectively, these findings suggest a role for CRLs and the ubiquitin proteasome system in suppression of p27 in SI-NET, and inhibition of neddylation as a putative therapeutic strategy in SI-NET.
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| 4. |
- Johansson, Henrik J., et al.
(author)
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Breast cancer quantitative proteome and proteogenomic landscape
- 2019
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In: Nature Communications. - : NATURE PUBLISHING GROUP. - 2041-1723. ; 10
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Journal article (peer-reviewed)abstract
- In the preceding decades, molecular characterization has revolutionized breast cancer (BC) research and therapeutic approaches. Presented herein, an unbiased analysis of breast tumor proteomes, inclusive of 9995 proteins quantified across all tumors, for the first time recapitulates BC subtypes. Additionally, poor-prognosis basal-like and luminal B tumors are further subdivided by immune component infiltration, suggesting the current classification is incomplete. Proteome-based networks distinguish functional protein modules for breast tumor groups, with co-expression of EGFR and MET marking ductal carcinoma in situ regions of normal-like tumors and lending to a more accurate classification of this poorly defined subtype. Genes included within prognostic mRNA panels have significantly higher than average mRNA-protein correlations, and gene copy number alterations are dampened at the protein-level; underscoring the value of proteome quantification for prognostication and phenotypic classification. Furthermore, protein products mapping to non-coding genomic regions are identified; highlighting a potential new class of tumor-specific immunotherapeutic targets.
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| 5. |
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| 6. |
- Lehtiö, Janne, et al.
(author)
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Proteogenomics of non-small cell lung cancer reveals molecular subtypes associated with specific therapeutic targets and immune-evasion mechanisms
- 2021
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In: Nature Cancer. - : Springer Science and Business Media LLC. - 2662-1347. ; 2:11, s. 1224-1242
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Journal article (peer-reviewed)abstract
- Despite major advancements in lung cancer treatment, long-term survival is still rare and a deeper understanding of molecular phenotypes would allow the identification of specific cancer dependencies and immune-evasion mechanisms. Here we performed in-depth mass-spectrometry-based proteogenomic analysis of 141 tumors representing all major histologies of non-small cell lung cancer (NSCLC). We identified six distinct proteome subtypes with striking differences in immune cell composition and subtype-specific expression of immune checkpoints. Unexpectedly, high neoantigen burden was linked to global hypomethylation and complex neoantigens mapped to genomic regions, such as endogenous retroviral elements and introns, in immune-cold subtypes. Further, we linked immune evasion with LAG-3 via STK11 mutation-dependent HNF1A activation and FGL1 expression. Finally, we develop a data-independent acquisition mass-spectrometry-based NSCLC subtype classification method, validate it in an independent cohort of 208 NSCLC cases and demonstrate its clinical utility by analyzing an additional cohort of 84 late-stage NSCLC biopsy samples.
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| 7. |
- Lewensohn, Rolf, et al.
(author)
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S100a6 and/or s100a4 inhibitors for treating cancer
- 2008
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Patent (pop. science, debate, etc.)abstract
- Aspects of this invention relate to the fields of molecular biology and medicine. More specifically, disclosed herein are several approaches to provide subjects suffering from cancer with an inhibitor of S100A6 and/or S 100A4 alone or in combination with other cancer therapies so as to improve the cancer therapy and/or more efficiently treat cancer, in particular forms of cancer that are resistant to other therapies. Also disclosed herein are approaches for using S 100A6 and/or S 100A4 as a biomarker for metastases. Further, disclosed herein are approaches for using S 100A6 and/or S 100A4 as a biomarker for cancer therapies, in particular, as a biomarker to determine individual responses to cancer therapies. In addition, disclosed herein are approaches to identifying S 100A6 and/or S 100A4 inhibitors, for example, that act synergistically with a cancer therapy.
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| 8. |
- Mou, Tian, et al.
(author)
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The transcriptome-wide landscape of molecular subtype-specific mRNA expression profiles in acute myeloid leukemia
- 2021
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In: American Journal of Hematology. - : John Wiley & Sons. - 0361-8609 .- 1096-8652. ; 96:5, s. 580-588
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Journal article (peer-reviewed)abstract
- Molecular classification of acute myeloid leukemia (AML) aids prognostic stratification and clinical management. Our aim in this study is to identify transcriptome-wide mRNAs that are specific to each of the molecular subtypes of AML. We analyzed RNA-sequencing data of 955 AML samples from three cohorts, including the BeatAML project, the Cancer Genome Atlas, and a cohort of Swedish patients to provide a comprehensive transcriptome-wide view of subtype-specific mRNA expression. We identified 729 subtype-specific mRNAs, discovered in the BeatAML project and validated in the other two cohorts. Using unique proteomics data, we also validated the presence of subtype-specific mRNAs at the protein level, yielding a rich collection of potential protein-based biomarkers for the AML community. To enable the exploration of subtype-specific mRNA expression by the broader scientific community, we provide an interactive resource to the public.
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| 9. |
- Orre, Lukas M., et al.
(author)
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FUNCTIONAL STUDIES OF S100A6 USING PROTEOMICS
- 2008
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In: Anticancer Research. - : INT INST ANTICANCER RESEARCH. - 0250-7005 .- 1791-7530. ; 28:5C, s. 3430-3431
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Journal article (other academic/artistic)
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| 10. |
- Orre, Lukas M., et al.
(author)
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p53 is involved in clearance of ionizing radiation-induced RAD51 foci in a human colon cancer cell line
- 2006
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In: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 342:4, s. 1211-7
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Journal article (peer-reviewed)abstract
- We have investigated p53-related differences in cellular response to DNA damaging agents, focusing on p53s effects on RAD51 protein level and sub-cellular localization post exposure to ionizing radiation. In a human colon cancer cell line, HCT116 and its isogenic p53-/- subcell line we show here p53-independent RAD51 foci formation but interestingly the resolution of RAD51 foci showed clear p53 dependence. In p53 wt cells, but not in p53-/- cells, RAD51 protein level decreased 48 h post irradiation and fluorescence immunostaining showed resolution of RAD51 foci and relocalization of RAD51 to nucleoli at time points corresponding to the decrease in RAD51 protein level. Both cell lines rejoined DNA double strand breaks efficiently with similar kinetics and p53 status did not influence sensitivity to DNA damaging agents. We suggest that p53 has a role in RAD51 clearance post DSB repair and that nucleoli might be sites of RAD51 protein degradation.
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| 11. |
- Orre, Lukas Minus, et al.
(author)
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SubCellBarCode : Proteome-wide Mapping of Protein Localization and Relocalization
- 2019
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In: Molecular Cell. - : Elsevier BV. - 1097-2765 .- 1097-4164. ; 73:1, s. 166-182.e7
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Journal article (peer-reviewed)abstract
- Subcellular localization is a main determinant of protein function; however, a global view of cellular proteome organization remains relatively unexplored. We have developed a robust mass spectrometry-based analysis pipeline to generate a proteome-wide view of subcellular localization for proteins mapping to 12,418 individual genes across five cell lines. Based on more than 83,000 unique classifications and correlation profiling, we investigate the effect of alternative splicing and protein domains on localization, complex member co-localization, cell-type-specific localization, as well as protein relocalization after growth factor inhibition. Our analysis provides information about the cellular architecture and complexity of the spatial organization of the proteome; we show that the majority of proteins have a single main subcellular location, that alternative splicing rarely affects subcellular location, and that cell types are best distinguished by expression of proteins exposed to the surrounding environment. The resource is freely accessible via www.subcellbarcode.org.
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| 12. |
- Orre, Lukas
(author)
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Proteomic analysis of DNA damage induced stress signaling with focus on p53 : S100A6 regulation, function and potential as biomarker in lung cancer and as a novel therapeutic target
- 2008
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Doctoral thesis (other academic/artistic)abstract
- In this thesis powerful proteomics methods were used to reveal novel proteins involved in the cellular response to DNA damaging treatment. The goal was to find proteins with potential as biomarkers for prediction of cancer prognosis and response to cancer therapy, but also to find potential novel targets of cancer therapy. In addition, the impact of the tumor suppressor p53 on the cellular response to DNA damaging treatment was investigated. When comparing two isogenic colon cancer cell lines (HCT116 p53wt and p53-/-) we were unable to detect p53 dependent differences in sensitivity to ionizing radiation (IR) or DNA damaging drugs. p53 did however alter the localization and reduce the abundance of Rad51, a key protein in homologous recombination repair, in response to IR. Our data thus indicate that p53 is involved in negative regulation of homologous recombination. The same cell line pair was also used in a proteomics time course study to identify novel proteins involved in the cellular response to DNA damaging agents (IR). In this study we discovered that the small calcium binding protein S100A6 was upregulated in a p53 dependent manner in irradiated cells. In addition to the upregulation of S100A6 post irradiation we also discovered that the post translational modification pattern and the sub cellular localization were altered. The biological functions of S100A6 were not known at the time of this discovery, but several studies had implicated S100A6 in carcinogenesis as it was shown upregulated in several different types of cancer. We therefore decided to pursue this finding and study the biological role of S100A6 in detail. The expression of S100A6 was investigated in stage I non-small cell lung cancer by immunohistochemistry using tissue microarrays. S100A6 was found overexpressed in cancer cells, and S100A6 expression correlated with wt p53 expression. S100A6 expression also correlated with patient survival in a subset of the cohort. In order to elucidate the function of S100A6 we set out to identify novel S100A6 interacting proteins. Using immunoprecipitation and proteomics methods we identified Ubiquilin-1 as a novel S100A6 interacting protein. Ubiquilin-1 is involved in regulationof proteasome mediated degradation of ubiquitinated proteins such as p53 and IkappaBa. siRNA mediated silencing of S100A6 resulted in stabilisation of both p53 and IkappaBa. Mass spectrometry based proteomics further revealed that S100A6 silencing reduced proteasomal processing of NFkappaB2 p100. S100A6 has earlier been suggested involved also in degradation of beta-catenin as S100A6 interacts with a component of the complex responsible for ubiquitination of beta-catenin. Using S100A6 silencing we were able to show increased degradation of beta-catenin. S100A6 silencing also increased the cellular sensitivity to ionizing radiation. In conclusion our data indicates that overexpression of S100A6 in cancer cells would result in a survival benefit through inhibition of apoptosis via increased p53 degradation and stimulation of proliferation via increased NFkappaB and beta-cateninsignaling. Our data also indicates that S100A6 is upregulated at later timepoints post irradiation to inhibit apoptosis and cell cycle arrest, allowing cells with repaired DNA damage to re-enter the cell cycle. These findings suggest the potential of S100A6 as a novel target of cancer therapy.
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| 13. |
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| 14. |
- Pernemalm, Maria, et al.
(author)
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Evaluation of three principally different intact protein prefractionation methods for plasma biomarker discovery.
- 2008
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In: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 7:7, s. 2712-2722
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Journal article (peer-reviewed)abstract
- The aim of this study was to evaluate three principally different top-down protein prefractionation methods for plasma: high-abundance protein depletion, size fractionation and peptide ligand affinity beads, focusing in particular on compatibility with downstream analysis, reproducibility and analytical depth. Our data clearly demonstrates the benefit of high-abundance protein depletion. However, MS/MS analysis of the proteins eluted from the high-abundance protein depletion column show that more proteins than aimed for are removed and, in addition, that the depletion efficacy varies between the different high-abundance proteins. Although a smaller number of proteins were identified per fraction using the peptide ligand affinity beads, this technique showed to be both robust and versatile. Size fractionation, as performed in this study, focusing on the low molecular weight proteome using a combination of gel filtration chromatography and molecular weight cutoff filters, showed limitations in the molecular weight cutoff precision leading detection of high molecular weight proteins and, in the case of the cutoff filters, high variability. GeLC-MS/MS analysis of the fractionation methods in combination with pathway analysis demonstrates that increased fractionation primarily leads to high proteome coverage of pathways related to biological functions of plasma, such as acute phase reaction, complement cascade and coagulation. Further, the prefractionation methods in this study induces limited effect on the proportion of tissue proteins detected, thereby highlighting the importance of extensive or targeted downstream fractionation.
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| 15. |
- Stranneheim, Henrik, et al.
(author)
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A comparison between protein profiles of B cell subpopulations and mantle cell lymphoma cells
- 2009
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In: Proteome Science. - : Springer Science and Business Media LLC. - 1477-5956. ; 7, s. 43-
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Journal article (peer-reviewed)abstract
- Background: B-cell lymphomas are thought to reflect different stages of B-cell maturation. Based on cytogenetics and molecular markers, mantle cell lymphoma (MCL) is presumed to derive predominantly from naive, pre-germinal centre (pre-GC) B lymphocytes. The aim of this study was to develop a method to investigate the similarity between MCL cells and different B-cell compartments on a protein expression level. Methods: Subpopulations of B cells representing the germinal centre (GC), the pre-GC mantle zone and the post-GC marginal zone were isolated from tonsils using automated magnetic cell sorting (AutoMACS) of cells based on their expression of CD27 and IgD. Protein profiling of the B cell subsets, of cell lines representing different lymphomas and of primary MCL samples was performed using top-down proteomics profiling by surface-enhanced laser detection/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Results: Quantitative MS data of significant protein peaks (p-value < 0.05) separating the three B-cell subpopulations were generated. Together, hierarchical clustering and principal component analysis (PCA) showed that the primary MCL samples clustered together with the pre- and post-GC subpopulations. Both primary MCL cells and MCL cell lines were clearly separated from the B cells representing the GC compartment. Conclusion: AutoMACS sorting generates sufficient purity to enable a comparison between protein profiles of B cell subpopulations and malignant B lymphocytes applying SELDI-TOF-MS. Further validation with an increased number of patient samples and identification of differentially expressed proteins would enable a search for possible treatment targets that are expressed during the early development of MCL.
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| 16. |
- Struyf, Nona, et al.
(author)
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Delineating functional and molecular impact of ex vivo sample handling in precision medicine
- 2024
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In: npj Precision Oncology. - : Springer Nature. - 2397-768X. ; 8:1
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Journal article (peer-reviewed)abstract
- Consistent handling of samples is crucial for achieving reproducible molecular and functional testing results in translational research. Here, we used 229 acute myeloid leukemia (AML) patient samples to assess the impact of sample handling on high-throughput functional drug testing, mass spectrometry-based proteomics, and flow cytometry. Our data revealed novel and previously described changes in cell phenotype and drug response dependent on sample biobanking. Specifically, myeloid cells with a CD117 (c-KIT) positive phenotype decreased after biobanking, potentially distorting cell population representations and affecting drugs targeting these cells. Additionally, highly granular AML cell numbers decreased after freezing. Secondly, protein expression levels, as well as sensitivity to drugs targeting cell proliferation, metabolism, tyrosine kinases (e.g., JAK, KIT, FLT3), and BH3 mimetics were notably affected by biobanking. Moreover, drug response profiles of paired fresh and frozen samples showed that freezing samples can lead to systematic errors in drug sensitivity scores. While a high correlation between fresh and frozen for the entire drug library was observed, freezing cells had a considerable impact at an individual level, which could influence outcomes in translational studies. Our study highlights conditions where standardization is needed to improve reproducibility, and where validation of data generated from biobanked cohorts may be particularly important.
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| 17. |
- Trac, Quang Thinh, et al.
(author)
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Prediction model for drug response of acute myeloid leukemia patients
- 2023
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In: npj Precision Oncology. - : Springer Nature. - 2397-768X. ; 7
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Journal article (peer-reviewed)abstract
- Despite some encouraging successes, predicting the therapy response of acute myeloid leukemia (AML) patients remains highly challenging due to tumor heterogeneity. Here we aim to develop and validate MDREAM, a robust ensemble-based prediction model for drug response in AML based on an integration of omics data, including mutations and gene expression, and large-scale drug testing. Briefly, MDREAM is first trained in the BeatAML cohort (n = 278), and then validated in the BeatAML (n = 183) and two external cohorts, including a Swedish AML cohort (n = 45) and a relapsed/refractory acute leukemia cohort (n = 12). The final prediction is based on 122 ensemble models, each corresponding to a drug. A confidence score metric is used to convey the uncertainty of predictions; among predictions with a confidence score >0.75, the validated proportion of good responders is 77%. The Spearman correlations between the predicted and the observed drug response are 0.68 (95% CI: [0.64, 0.68]) in the BeatAML validation set, -0.49 (95% CI: [-0.53, -0.44]) in the Swedish cohort and 0.59 (95% CI: [0.51, 0.67]) in the relapsed/refractory cohort. A web-based implementation of MDREAM is publicly available at https://www.meb.ki.se/shiny/truvu/MDREAM/.
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| 18. |
- Veerman, Rosanne E., et al.
(author)
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Molecular evaluation of five different isolation methods for extracellular vesicles reveals different clinical applicability and subcellular origin
- 2021
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In: Journal of Extracellular Vesicles. - : Wiley. - 2001-3078. ; 10:9
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Journal article (peer-reviewed)abstract
- Extracellular vesicles (EVs) are increasingly tested as therapeutic vehicles and biomarkers, but still EV subtypes are not fully characterised. To isolate EVs with few co-isolated entities, a combination of methods is needed. However, this is time-consuming and requires large sample volumes, often not feasible in most clinical studies or in studies where small sample volumes are available. Therefore, we compared EVs rendered by five commonly used methods based on different principles from conditioned cell medium and 250 mu l or 3 ml plasma, that is, precipitation (ExoQuick ULTRA), membrane affinity (exoEasy Maxi Kit), size-exclusion chromatography (qEVoriginal), iodixanol gradient (OptiPrep), and phosphatidylserine affinity (MagCapture). EVs were characterised by electron microscopy, Nanoparticle Tracking Analysis, Bioanalyzer, flow cytometry, and LC-MS/MS. The different methods yielded samples of different morphology, particle size, and proteomic profile. For the conditioned medium, Izon 35 isolated the highest number of EV proteins followed by exoEasy, which also isolated fewer non-EV proteins. For the plasma samples, exoEasy isolated a high number of EV proteins and few non-EV proteins, while Izon 70 isolated the most EV proteins. We conclude that no method is perfect for all studies, rather, different methods are suited depending on sample type and interest in EV subtype, in addition to sample volume and budget.
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| 19. |
- Zhu, Yafeng, et al.
(author)
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Discovery of coding regions in the human genome by integrated proteogenomics analysis workflow
- 2018
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In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 9
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Journal article (peer-reviewed)abstract
- Proteogenomics enable the discovery of novel peptides (from unannotated genomic protein-coding loci) and single amino acid variant peptides (derived from single-nucleotide polymorphisms and mutations). Increasing the reliability of these identifications is crucial to ensure their usefulness for genome annotation and potential application as neoantigens in cancer immunotherapy. We here present integrated proteogenomics analysis workflow (IPAW), which combines peptide discovery, curation, and validation. IPAW includes the SpectrumAI tool for automated inspection of MS/MS spectra, eliminating false identifications of single-residue substitution peptides. We employ IPAW to analyze two proteomics data sets acquired from A431 cells and five normal human tissues using extended (pH range, 3-10) high-resolution isoelectric focusing (HiRIEF) pre-fractionation and TMT-based peptide quantitation. The IPAW results provide evidence for the translation of pseudogenes, lncRNAs, short ORFs, alternative ORFs, N-terminal extensions, and intronic sequences. Moreover, our quantitative analysis indicates that protein production from certain pseudogenes and lncRNAs is tissue specific.
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