| 1. |
- Arvidsson, Yvonne, 1960, et al.
(författare)
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miRNA profiling of small intestinal neuroendocrine tumors defines novel molecular subtypes and identifies miR-375 as a biomarker of patient survival
- 2018
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Ingår i: Modern Pathology. - : Elsevier BV. - 0893-3952 .- 1530-0285. ; 31, s. 1302-1317
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Tidskriftsartikel (refereegranskat)abstract
- © 2018, United States & Canadian Academy of Pathology. The aim of this study was to define the miRNA profile of small intestinal neuroendocrine tumors and to search for novel molecular subgroups and prognostic biomarkers. miRNA profiling was conducted on 42 tumors from 37 patients who underwent surgery for small intestinal neuroendocrine tumors. Unsupervised hierarchical clustering analysis of miRNA profiles identified two groups of tumor metastases, denoted cluster M1 and M2. The smaller cluster M1 was associated with shorter overall survival and contained tumors with higher grade (WHO grade G2/3) and multiple chromosomal gains including gain of chromosome 14. Tumors of cluster M1 had elevated expression of miR-1246 and miR-663a, and reduced levels of miR-488-3p. Pathway analysis predicted Wnt signaling to be the most significantly altered signaling pathway between clusters M1 and M2. Analysis of miRNA expression in relation to tumor proliferation rate showed significant alterations including downregulation of miR-137 and miR-204-5p in tumors with Ki67 index above 3%. Similarly, tumor progression was associated with significant alterations in miRNA expression, e.g. higher expression of miR-95 and miR-210, and lower expression of miR-378a-3p in metastases. Pathway analysis predicted Wnt signaling to be altered during tumor progression, which was supported by decreased nuclear translocation of β-catenin in metastases. Survival analysis revealed that downregulation of miR-375 was associated with shorter overall survival. We performed in situ hybridization on biopsies from an independent cohort of small intestinal neuroendocrine tumors using tissue microarrays. Expression of miR-375 was found in 578/635 (91%) biopsies and survival analysis confirmed that there was a correlation between downregulation of miR-375 in tumor metastases and shorter patient survival. We conclude that miRNA profiling defines novel molecular subgroups of metastatic small intestinal neuroendocrine tumors and identifies miRNAs associated with tumor proliferation rate and progression. miR-375 is highly expressed in small intestinal neuroendocrine tumors and may be used as a prognostic biomarker.
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| 2. |
- Bengtsson-Palme, Johan, 1985, et al.
(författare)
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Strategies to improve usability and preserve accuracy in biological sequence databases
- 2016
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Ingår i: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861. ; 16:18, s. 2454-2460
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Tidskriftsartikel (refereegranskat)abstract
- Biology is increasingly dependent on large-scale analysis, such as proteomics, creating a requirement for efficient bioinformatics. Bioinformatic predictions of biological functions rely upon correctly annotated database sequences, and the presence of inaccurately annotated or otherwise poorly described sequences introduces noise and bias to biological analyses. Accurate annotations are, for example, pivotal for correct identifications of polypeptide fragments. However, standards for how sequence databases are organized and presented are currently insufficient. Here, we propose five strategies to address fundamental issues in the annotation of sequence databases: (i) to clearly separate experimentally verified and unverified sequence entries; (ii) to enable a system for tracing the origins of annotations; (iii) to separate entries with high-quality, informative annotation from less useful ones; (iv) to integrate automated quality-control software whenever such tools exist; and (v) to facilitate post-submission editing of annotations and metadata associated with sequences. We believe that implementation of these strategies, for example as requirements for publication of database papers, would enable biology to better take advantage of large-scale data.
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| 3. |
- Hofving, Tobias, 1989, et al.
(författare)
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SMAD4 haploinsufficiency in small intestinal neuroendocrine tumors
- 2021
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Ingår i: BMC Cancer. - : Springer Science and Business Media LLC. - 1471-2407. ; 21:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Patients with small intestinal neuroendocrine tumors (SINETs) frequently present with lymph node and liver metastases at the time of diagnosis, but the molecular changes that lead to the progression of these tumors are largely unknown. Sequencing studies have only identified recurrent point mutations at low frequencies with CDKN1B being the most common harboring heterozygous mutations in less than 10% of all tumors. Although SINETs are genetically stable tumors with a low frequency of point mutations and indels, they often harbor recurrent hemizygous copy number alterations (CNAs) yet the functional implications of these CNA are unclear. Methods: Utilizing comparative genomic hybridization (CGH) arrays we analyzed the CNA profile of 131 SINETs from 117 patients. Two tumor suppressor genes and corresponding proteins i.e. SMAD4, and CDKN1B, were further characterized using a tissue microarray (TMA) with 846 SINETs. Immunohistochemistry (IHC) was used to quantify protein expression in TMA samples and this was correlated with chromosome number evaluated with fluorescent in-situ hybridization (FISH). Intestinal tissue from a Smad4+/− mouse model was used to detect entero-endocrine cell hyperplasia with IHC. Results: Analyzing the CGH arrays we found loss of chromosome 18q and SMAD4 in 71% of SINETs and that focal loss of chromosome 12 affecting the CDKN1B was present in 9.4% of SINETs. No homozygous loss of chromosome 18 was detected. Hemizygous loss of SMAD4, but not CDKN1B, significantly correlated with reduced protein levels but hemizygous loss of SMAD4 did not induce entero-endocrine cell hyperplasia in the Smad4+/− mouse model. In addition, patients with low SMAD4 protein expression in primary tumors more often presented with metastatic disease. Conclusions: Hemizygous loss of chromosome 18q and the SMAD4 gene is the most common genetic event in SINETs and our results suggests that this could influence SMAD4 protein expression and spread of metastases. Although SMAD4 haploinsufficiency alone did not induce tumor initiation, loss of chromosome 18 could represent an evolutionary advantage in SINETs explaining the high prevalence of this aberration. Functional consequences of reduced SMAD4 protein levels could hypothetically be a potential mechanism as to why loss of chromosome 18 appears to be clonally selected in SINETs.
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| 4. |
- Juul-Dam, KL, et al.
(författare)
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Deep Sequencing of Leukemia-specific Mutations in Peripheral Blood Identifies Children with Imminent Relapse of Acute Myeloid Leukemia
- 2019
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Ingår i: NOPHO Annual Meeting 2019.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Relapse remains the major problem in childhood acute myeloid leukemia (AML). The outcome in children with imminent relapse may improve if preemptive therapy is initiated at first evidence of leukemia regrowth. This requires measurable residual disease (MRD) monitoring after therapy completion, but only 40% of children with AML harbor genetic abnormalities applicable for quantification using standardized qPCR assays. To enable disease surveillance for all patients, we investigated the potential of early relapse detection in peripheral blood (PB) using patient-tailored deep sequencing (DS) MRD analysis. PB samples were collected at monthly intervals during follow-up from 45 children diagnosed with AML and treated according to the NOPHO-DBH AML 2012 protocol between January 2013 and May 2016 in Denmark, Norway, Sweden and Finland (508 samples, median 11 samples/patient, range 3–27). In relapsed patients, MRD-suitable leukemia-specific single nucleotide variants (SNVs) were identified with exome sequencing (ES) in diagnostic samples and verified at relapse. SNVs were analyzed in PB samples obtained during the months before overt relapse using DS with a sensitivity of 0.02% variant allele frequency (VAF). Until October 1st 2017, 14 patients experienced relapse within 18 months from therapy completion, and in 6 patients analysis has been completed. ES identified 37 leukemia-specific SNVs at diagnosis (median 4 SNVs/patient, range 2–12) of which 23 were also present at relapse (median 3 SNVs/patient, range 1–9). Fourteen MRD-suitable SNVs (1–3/patient) were quantified with DS. In all patients, at least one SNV was detected in PB before overt relapse occurred. The first PB sample showing MRD positivity (median error corrected VAF 0.15%, range 0.03–0.85) preceded hematological relapse at a median interval of 2.3 months (range 0.6–4.7). In conclusion, high-sensitivity quantification of leukemia-specific SNVs can facilitate early detection of imminent relapse and provide a chance for initiation of preemptive treatment.
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| 5. |
- Juul-Dam, KL, et al.
(författare)
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Patient-Tailored Deep Sequencing of Peripheral Blood Enables Early Detection of Relapse in Childhood Acute Myeloid Leukemia
- 2019
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Ingår i: Blood Vpl 134 Suppl 1 1456. - : American Society of Hematology. - 0006-4971 .- 1528-0020.
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Konferensbidrag (refereegranskat)abstract
- Relapse remains a major therapeutic challenge in children with acute myeloid leukemia (AML). Outcome after relapse may improve if preemptive therapy is initiated at first evidence of leukemia regrowth. Early detection of imminent relapse requires molecular measurable residual disease (MRD) monitoring after therapy completion. Today, this is possible only in about 40% of children with AML that harbor genetic abnormalities applicable for quantification using standardized qPCR assays. To enable disease surveillance for all patients, we developed patient-tailored deep sequencing (DS) MRD analysis, which provides highly sensitive detection of leukemia-specific mutations. We investigated the potential of this method for early relapse detection in peripheral blood (PB), the only easily accessible source for MRD sampling in children. PB samples were collected at monthly intervals during follow-up from 45 children diagnosed with AML and treated according to The Nordic Society of Pediatric Haematology and Oncology (NOPHO)-DBH AML 2012 protocol between January 2013 and May 2016 in Denmark, Norway, Sweden and Finland (508 samples, median 11 samples/patient, range 3-27). Nine patients with relapse (median age 5 years, range 0-8) had available diagnostic and relapse material and were included in this study. The patients displayed core binding factor abnormalities (n=3), KMT2A-rearrangements (n=3), monosomy 7 (n=1) or normal karyotype (n=2) at AML diagnosis. Leukemia-specific single nucleotide variants (SNVs) were identified with exome sequencing (ES) of sorted leukemic cells with lymphocytes or remission PB as constitutive DNA template. A variant allele frequency (VAF) with 95% confidence interval including 50% indicates presence of the mutation in all leukemic cells at diagnosis. With the exception of 2 cases with only subclonal mutations at diagnosis, leukemia-specific SNVs with VAF of 50% at diagnosis and persistence at relapse were selected as MRD targets. MRD target mutations were quantified in PB samples preceding overt relapse using patient-tailored DS assays with sensitivity of VAF 0.02%. In diagnostic samples, ES identified 53 leukemia-specific SNVs (median 4 SNVs/patient, range 2-12) of which 33 were also present at relapse (median 2 SNVs/patient, range 1-9). The number of mutations identified at diagnosis increased with age (Rs 0.83, p=0.006). All patients had at least one leukemia-specific SNV detected at both diagnosis and relapse. Twenty-one MRD target mutations (median 2 SNVs/patient, range 1-3) were quantified in PB (55 samples, median sampling interval 28 days, range 11-80) using DS. In 8/9 patients, at least one SNV was detected in PB before overt relapse occurred. The first PB sample showing MRD positivity (median VAF 0.14%, range 0.03-0.44) preceded hematological relapse at a median interval of 3 months (range 0-7.9). In 6 patients not preemptively treated, the median doubling time based on VAF increments was 7 days, with great variability between individuals and genotypes (range 4-28 days). Three patients had molecular relapse diagnosed by qPCR used in clinical diagnostics and received individualized preemptive treatment. In these 3 patients, DS detected mutations in PB for >100 days preceding overt relapse and the doubling times were 14, 25 and 36 days. In conclusion, DS of leukemia-specific mutations at frequent intervals in PB enables early detection of relapse and ES at diagnosis may identify SNVs applicable for such longitudinal MRD monitoring. This approach facilitates molecular disease surveillance and initiation of preemptive therapy in AML patients without established qPCR targets.
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| 6. |
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| 7. |
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| 8. |
- Malmberg, Erik, et al.
(författare)
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Patient-tailored analysis of minimal residual disease in acute myeloid leukemia using next-generation sequencing
- 2017
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Ingår i: European Journal of Haematology. - : Wiley. - 0902-4441 .- 1600-0609. ; 98:1, s. 26-37
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Tidskriftsartikel (refereegranskat)abstract
- Next-generation sequencing techniques have revealed that leukemic cells in acute myeloid leukemia often are characterized by a limited number of somatic mutations. These mutations can be the basis for the detection of leukemic cells in follow-up samples. The aim of this study was to identify leukemia-specific mutations in cells from patients with acute myeloid leukemia and to use these mutations as markers for minimal residual disease. Leukemic cells and normal lymphocytes were simultaneously isolated at diagnosis from 17 patients with acute myeloid leukemia using fluorescence-activated cell sorting. Exome sequencing of these cells identified 240 leukemia-specific single nucleotide variations and 22 small insertions and deletions. Based on estimated allele frequencies and their accuracies, 191 of these mutations qualified as candidates for minimal residual disease analysis. Targeted deep sequencing with a significance threshold of 0.027% for single nucleotide variations and 0.006% for NPM1 type A mutation was developed for quantification of minimal residual disease. When tested on follow-up samples from a patient with acute myeloid leukemia, targeted deep sequencing of single nucleotide variations as well as NPM1 was more sensitive than minimal residual disease quantification with multiparameter flow cytometry. In conclusion, we here describe how exome sequencing can be used for identification of leukemia-specific mutations in samples already at diagnosis of acute myeloid leukemia. We also show that targeted deep sequencing of such mutations, including single nucleotide variations, can be used for high-sensitivity quantification of minimal residual disease in a patient-tailored manner.
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| 9. |
- Tomic, Tajana Tesan, et al.
(författare)
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MYO5B mutations in pheochromocytoma/paraganglioma promote cancer progression
- 2020
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Ingår i: PLOS Genetics. - : Public Library of Science. - 1553-7390 .- 1553-7404. ; 16:6
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Tidskriftsartikel (refereegranskat)abstract
- Identification of additional cancer-associated genes and secondary mutations driving the metastatic progression in pheochromocytoma and paraganglioma (PPGL) is important for subtyping, and may provide optimization of therapeutic regimens. We recently reported novel recurrent nonsynonymous mutations in the MYO5B gene in metastatic PPGL. Here, we explored the functional impact of these MYO5B mutations, and analyzed MYO5B expression in primary PPGL tumor cases in relation to mutation status. Immunohistochemistry and mRNA expression analysis in 30 PPGL tumors revealed an increased MYO5B expression in metastatic compared to non-metastatic cases. In addition, subcellular localization of MYO5B protein was altered from cytoplasmic to membranous in some metastatic tumors, and the strongest and most abnormal expression pattern was observed in a paraganglioma harboring a somatic MYO5B:p.G1611S mutation. In addition to five previously discovered MYO5B mutations, the present study of 30 PPGL (8 previous and 22 new samples) also revealed two, and hence recurrent, mutations in the gene paralog MYO5A. The three MYO5B missense mutations with the highest prediction scores (p.L587P, p.G1611S and p.R1641C) were selected and functionally validated using site directed mutagenesis and stable transfection into human neuroblastoma cells (SK-N-AS) and embryonic kidney cells (HEK293). In vitro analysis showed a significant increased proliferation rate in all three MYO5B mutated clones. The two somatically derived mutations, p.L587P and p.G1611S, were also found to increase the migration rate. Expression analysis of MYO5B mutants compared to wild type clones, demonstrated a significant enrichment of genes involved in migration, proliferation, cell adhesion, glucose metabolism, and cellular homeostasis. Our study validates the functional role of novel MYO5B mutations in proliferation and migration, and suggest the MYO5-pathway to be involved in the malignant progression in some PPGL tumors. © 2020 Tomic et al.
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| 10. |
- Wilzén, Annica, et al.
(författare)
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Malignant pheochromocytomas/paragangliomas harbor mutations in transport and cell adhesion genes.
- 2016
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Ingår i: International Journal of Cancer. - : Wiley. - 0020-7136 .- 1097-0215. ; 138:9, s. 2201-11
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Tidskriftsartikel (refereegranskat)abstract
- One out of ten patients with pheochromocytoma (PCC) and paraganglioma (PGL) develop malignant disease. Today there are no reliable pathological methods to predict malignancy at the time of diagnosis. Tumors harboring mutations in the succinate dehydrogenase subunit B (SDHB) gene often metastasize but the sequential genetic events resulting in malignant progression are not fully understood. The aim of this study was to identify somatic mutations that contribute to the malignant transformation of PCC/PGL. We performed pair-wise (tumor-normal) whole-exome sequencing to analyze the somatic mutational landscape in five malignant and four benign primary PCC/sympathetic PGL (sPGL), including two biological replicates from each specimen. In total, 225 unique somatic mutations were identified in 215 genes, with an average mutation rate of 0.54 mutations/megabase. Malignant tumors had a significantly higher number of mutations compared to benign tumors (p
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