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1.
Int J Mol Sci ; 23(9)2022 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-35563565

RESUMO

Somatic loss of function mutations in cohesin genes are frequently associated with various cancer types, while cohesin disruption in the germline causes cohesinopathies such as Cornelia-de-Lange syndrome (CdLS). Here, we present the discovery of a recurrent heterozygous RAD21 germline aberration at amino acid position 298 (p.P298S/A) identified in three children with lymphoblastic leukemia or lymphoma in a total dataset of 482 pediatric cancer patients. While RAD21 p.P298S/A did not disrupt the formation of the cohesin complex, it altered RAD21 gene expression, DNA damage response and primary patient fibroblasts showed increased G2/M arrest after irradiation and Mitomycin-C treatment. Subsequent single-cell RNA-sequencing analysis of healthy human bone marrow confirmed the upregulation of distinct cohesin gene patterns during hematopoiesis, highlighting the importance of RAD21 expression within proliferating B- and T-cells. Our clinical and functional data therefore suggest that RAD21 germline variants can predispose to childhood lymphoblastic leukemia or lymphoma without displaying a CdLS phenotype.


Assuntos
Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Linfoma , Leucemia-Linfoma Linfoblástico de Células Precursoras , Apoptose , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Criança , Proteínas de Ligação a DNA/genética , Síndrome de Cornélia de Lange/genética , Pontos de Checagem da Fase G2 do Ciclo Celular , Células Germinativas/metabolismo , Humanos , Linfoma/genética , Mutação , Fenótipo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética
2.
Int J Cancer ; 148(6): 1438-1451, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-32949162

RESUMO

DNA sequencing and RNA sequencing are increasingly applied in precision oncology, where molecular tumor boards evaluate the actionability of genetic events in individual tumors to guide targeted treatment. To work toward an additional level of patient characterization, we assessed the abundance and activity of 27 proteins in 134 patients whose tumors had previously undergone whole-exome and RNA sequencing within the Molecularly Aided Stratification for Tumor Eradication Research (MASTER) program of National Center for Tumor Diseases, Heidelberg. Proteomic and phosphoproteomic targets were selected to reflect the most relevant therapeutic baskets in MASTER. Among six different therapeutic baskets, the proteomic data supported treatment recommendations that were based on DNA and RNA analyses in 10% to 57% and frequently suggested alternative treatment options. In several cases, protein activities explained the patients' clinical course and provided potential explanations for treatment failure. Our study indicates that the integrative analysis of DNA, RNA and protein data may refine therapeutic stratification of individual patients and, thus, holds potential to increase the success rate of precision cancer therapy. Prospective validation studies are needed to advance the integration of proteomic analysis into precision oncology.


Assuntos
Oncologia/métodos , Terapia de Alvo Molecular/métodos , Neoplasias , Medicina de Precisão/métodos , Proteômica/métodos , Adulto , Idoso , Biomarcadores Tumorais/análise , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/genética , Neoplasias/terapia , Estudo de Prova de Conceito
3.
Int J Cancer ; 145(12): 3299-3310, 2019 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-31135957

RESUMO

Genomic sequencing projects unraveled the mutational landscape of head and neck squamous cell carcinoma (HNSCC) and provided a comprehensive catalog of somatic mutations. However, the limited number of significant cancer-related genes obtained so far only partially explains the biological complexity of HNSCC and hampers the development of novel diagnostic biomarkers and therapeutic targets. We pursued a multiscale omics approach based on whole-exome sequencing, global DNA methylation and gene expression profiling data derived from tumor samples of the HIPO-HNC cohort (n = 87), and confirmed new findings with datasets from The Cancer Genome Atlas (TCGA). Promoter methylation was confirmed by MassARRAY analysis and protein expression was assessed by immunohistochemistry and immunofluorescence staining. We discovered a set of cancer-related genes with frequent somatic mutations and high frequency of promoter methylation. This included the ryanodine receptor 2 (RYR2), which showed variable promoter methylation and expression in both tumor samples and cell lines. Immunohistochemical staining of tissue sections unraveled a gradual loss of RYR2 expression from normal mucosa via dysplastic lesion to invasive cancer and indicated that reduced RYR2 expression in adjacent tissue and precancerous lesions might serve as risk factor for unfavorable prognosis and upcoming malignant conversion. In summary, our data indicate that impaired RYR2 function by either somatic mutation or epigenetic silencing is a common event in HNSCC pathogenesis. Detection of RYR2 expression and/or promoter methylation might enable risk assessment for malignant conversion of dysplastic lesions.


Assuntos
Metilação de DNA/genética , Neoplasias de Cabeça e Pescoço/genética , Mutação/genética , Regiões Promotoras Genéticas/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Estudos de Coortes , Ilhas de CpG/genética , Epigênese Genética/genética , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética
4.
Diabetologia ; 61(3): 641-657, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29185012

RESUMO

AIMS/HYPOTHESIS: Pancreatic islet beta cell failure causes type 2 diabetes in humans. To identify transcriptomic changes in type 2 diabetic islets, the Innovative Medicines Initiative for Diabetes: Improving beta-cell function and identification of diagnostic biomarkers for treatment monitoring in Diabetes (IMIDIA) consortium ( www.imidia.org ) established a comprehensive, unique multicentre biobank of human islets and pancreas tissues from organ donors and metabolically phenotyped pancreatectomised patients (PPP). METHODS: Affymetrix microarrays were used to assess the islet transcriptome of islets isolated either by enzymatic digestion from 103 organ donors (OD), including 84 non-diabetic and 19 type 2 diabetic individuals, or by laser capture microdissection (LCM) from surgical specimens of 103 PPP, including 32 non-diabetic, 36 with type 2 diabetes, 15 with impaired glucose tolerance (IGT) and 20 with recent-onset diabetes (<1 year), conceivably secondary to the pancreatic disorder leading to surgery (type 3c diabetes). Bioinformatics tools were used to (1) compare the islet transcriptome of type 2 diabetic vs non-diabetic OD and PPP as well as vs IGT and type 3c diabetes within the PPP group; and (2) identify transcription factors driving gene co-expression modules correlated with insulin secretion ex vivo and glucose tolerance in vivo. Selected genes of interest were validated for their expression and function in beta cells. RESULTS: Comparative transcriptomic analysis identified 19 genes differentially expressed (false discovery rate ≤0.05, fold change ≥1.5) in type 2 diabetic vs non-diabetic islets from OD and PPP. Nine out of these 19 dysregulated genes were not previously reported to be dysregulated in type 2 diabetic islets. Signature genes included TMEM37, which inhibited Ca2+-influx and insulin secretion in beta cells, and ARG2 and PPP1R1A, which promoted insulin secretion. Systems biology approaches identified HNF1A, PDX1 and REST as drivers of gene co-expression modules correlated with impaired insulin secretion or glucose tolerance, and 14 out of 19 differentially expressed type 2 diabetic islet signature genes were enriched in these modules. None of these signature genes was significantly dysregulated in islets of PPP with impaired glucose tolerance or type 3c diabetes. CONCLUSIONS/INTERPRETATION: These studies enabled the stringent definition of a novel transcriptomic signature of type 2 diabetic islets, regardless of islet source and isolation procedure. Lack of this signature in islets from PPP with IGT or type 3c diabetes indicates differences possibly due to peculiarities of these hyperglycaemic conditions and/or a role for duration and severity of hyperglycaemia. Alternatively, these transcriptomic changes capture, but may not precede, beta cell failure.


Assuntos
Bancos de Espécimes Biológicos , Diabetes Mellitus Tipo 2/metabolismo , Biologia de Sistemas/métodos , Doadores de Tecidos , Transcriptoma/genética , Idoso , Idoso de 80 Anos ou mais , Biologia Computacional , Feminino , Humanos , Masculino , Pancreatectomia
5.
Int J Cancer ; 141(5): 877-886, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28597939

RESUMO

Precision oncology implies the ability to predict which patients will likely respond to specific cancer therapies based on increasingly accurate, high-resolution molecular diagnostics as well as the functional and mechanistic understanding of individual tumors. While molecular stratification of patients can be achieved through different means, a promising approach is next-generation sequencing of tumor DNA and RNA, which can reveal genomic alterations that have immediate clinical implications. Furthermore, certain genetic alterations are shared across multiple histologic entities, raising the fundamental question of whether tumors should be treated by molecular profile and not tissue of origin. We here describe MASTER (Molecularly Aided Stratification for Tumor Eradication Research), a clinically applicable platform for prospective, biology-driven stratification of younger adults with advanced-stage cancer across all histologies and patients with rare tumors. We illustrate how a standardized workflow for selection and consenting of patients, sample processing, whole-exome/genome and RNA sequencing, bioinformatic analysis, rigorous validation of potentially actionable findings, and data evaluation by a dedicated molecular tumor board enables categorization of patients into different intervention baskets and formulation of evidence-based recommendations for clinical management. Critical next steps will be to increase the number of patients that can be offered comprehensive molecular analysis through collaborations and partnering, to explore ways in which additional technologies can aid in patient stratification and individualization of treatment, to stimulate clinically guided exploratory research projects, and to gradually move away from assessing the therapeutic activity of targeted interventions on a case-by-case basis toward controlled clinical trials of genomics-guided treatments.


Assuntos
Perfilação da Expressão Gênica/métodos , Oncologia/métodos , Neoplasias/genética , Medicina de Precisão/métodos , Humanos , Neoplasias/classificação
6.
Blood ; 126(8): 1005-8, 2015 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-26065650

RESUMO

Hairy cell leukemia (HCL) is marked by near 100% mutational frequency of BRAFV600E mutations. Recurrent cooperating genetic events that may contribute to HCL pathogenesis or affect the clinical course of HCL are currently not described. Therefore, we performed whole exome sequencing to explore the mutational landscape of purine analog refractory HCL. In addition to the disease-defining BRAFV600E mutations, we identified mutations in EZH2, ARID1A, and recurrent inactivating mutations of the cell cycle inhibitor CDKN1B (p27). Targeted deep sequencing of CDKN1B in a larger cohort of HCL patients identify deleterious CDKN1B mutations in 16% of patients with HCL (n = 13 of 81). In 11 of 13 patients the CDKN1B mutation was clonal, implying an early role of CDKN1B mutations in the pathogenesis of HCL. CDKN1B mutations were not found to impact clinical characteristics or outcome in this cohort. These data identify HCL as having the highest frequency of CDKN1B mutations among cancers and identify CDNK1B as the second most common mutated gene in HCL. Moreover, given the known function of CDNK1B, these data suggest a novel role for alterations in regulation of cell cycle and senescence in HCL with CDKN1B mutations.


Assuntos
Inibidor de Quinase Dependente de Ciclina p27/genética , Leucemia de Células Pilosas/genética , Mutação , Análise Mutacional de DNA , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Recidiva
8.
BMC Med Inform Decis Mak ; 13: 75, 2013 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-23883409

RESUMO

BACKGROUND: The usage of patient data for research poses risks concerning the patients' privacy and informational self-determination. Next-generation-sequencing technologies and various other methods gain data from biospecimen, both for translational research and personalized medicine. If these biospecimen are anonymized, individual research results from genomic research, which should be offered to patients in a clinically relevant timeframe, cannot be associated back to the individual. This raises an ethical concern and challenges the legitimacy of anonymized patient samples. In this paper we present a new approach which supports both data privacy and the possibility to give feedback to patients about their individual research results. METHODS: We examined previously published privacy concepts regarding a streamlined de-pseudonymization process and a patient-based pseudonym as applicable to research with genomic data and warehousing approaches. All concepts identified in the literature review were compared to each other and analyzed for their applicability to translational research projects. We evaluated how these concepts cope with challenges implicated by personalized medicine. Therefore, both person-centricity issues and a separation of pseudonymization and de-pseudonymization stood out as a central theme in our examination. This motivated us to enhance an existing pseudonymization method regarding a separation of duties. RESULTS: The existing concepts rely on external trusted third parties, making de-pseudonymization a multistage process involving additional interpersonal communication, which might cause critical delays in patient care. Therefore we propose an enhanced method with an asymmetric encryption scheme separating the duties of pseudonymization and de-pseudonymization. The pseudonymization service provider is unable to conclude the patient identifier from the pseudonym, but assigns this ability to an authorized third party (ombudsman) instead. To solve person-centricity issues, a collision-resistant function is incorporated into the method. These two facts combined enable us to address essential challenges in translational research. A productive software prototype was implemented to prove the functionality of the suggested translational, data privacy-preserving method. Eventually, we performed a threat analysis to evaluate potential hazards connected with this pseudonymization method. CONCLUSIONS: The proposed method offers sustainable organizational simplification regarding an ethically indicated, but secure and controlled process of de-pseudonymizing patients. A pseudonym is patient-centered to allow correlating separate datasets from one patient. Therefore, this method bridges the gap between bench and bedside in translational research while preserving patient privacy. Assigned ombudsmen are able to de-pseudonymize a patient, if an individual research result is clinically relevant.


Assuntos
Anônimos e Pseudônimos , Segurança Computacional , Sistemas de Identificação de Pacientes/métodos , Privacidade , Pesquisa Translacional Biomédica , Confidencialidade/normas , Genética Médica/métodos , Genética Médica/normas , Genômica/ética , Genômica/normas , Humanos , Armazenamento e Recuperação da Informação/métodos , Armazenamento e Recuperação da Informação/normas , Privacidade/legislação & jurisprudência , Avaliação da Tecnologia Biomédica , Simplificação do Trabalho
9.
Cell Rep Med ; 4(10): 101200, 2023 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-37734378

RESUMO

Targeted therapies are effective in treating cancer, but success depends on identifying cancer vulnerabilities. In our study, we utilize small RNA sequencing to examine the impact of pathway activation on microRNA (miRNA) expression patterns. Interestingly, we discover that miRNAs capable of inhibiting key members of activated pathways are frequently diminished. Building on this observation, we develop an approach that integrates a low-miRNA-expression signature to identify druggable target genes in cancer. We train and validate our approach in colorectal cancer cells and extend it to diverse cancer models using patient-derived in vitro and in vivo systems. Finally, we demonstrate its additional value to support genomic and transcriptomic-based drug prediction strategies in a pan-cancer patient cohort from the National Center for Tumor Diseases (NCT)/German Cancer Consortium (DKTK) Molecularly Aided Stratification for Tumor Eradication (MASTER) precision oncology trial. In conclusion, our strategy can predict cancer vulnerabilities with high sensitivity and accuracy and might be suitable for future therapy recommendations in a variety of cancer subtypes.


Assuntos
MicroRNAs , Neoplasias , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Medicina de Precisão , Genômica , Transcriptoma
10.
Proteomics ; 12(18): 2843-51, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22848051

RESUMO

Malted barley is an important ingredient used in the brewing and distilling industry worldwide. In this study, we used a proteomics approach to investigate the biochemical function of previously identified quantitative trait loci (QTLs) on barley chromosomes 1H and 4H that influence malting quality. Using a subset of barley introgression lines containing wild barley (Hordeum vulgare ssp. spontaneum) alleles at these QTLs, we validated that wild barley alleles at the chromosome 1H QTL reduced overall malting quality, whereas wild barley alleles at the chromosome 4H QTL improved the malting quality parameters α-amylase activity, VZ45, and Kolbach index compared to the control genotype Scarlett. 2DE was used to detect changes in protein expression during the first 72 h of micromalting associated with these QTLs. In total, 16 protein spots showed a significant change in expression between the introgression lines and Scarlett, of which 14 were successfully identified with MS. Notably, the wild barley alleles in the line containing the chromosome 4H QTL showed a sixfold increased expression of a limit dextrinase inhibitor. The possible role of the identified proteins in malting quality is discussed. The knowledge gained will assist ongoing research toward cloning the genes underlying these important QTL.


Assuntos
Hordeum/genética , Hordeum/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Cromossomos de Plantas/genética , Eletroforese em Gel Bidimensional , Regulação da Expressão Gênica de Plantas , Espectrometria de Massas , Proteômica , Locos de Características Quantitativas
11.
Nat Commun ; 13(1): 4485, 2022 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-35918329

RESUMO

The benefit of molecularly-informed therapies in cancer of unknown primary (CUP) is unclear. Here, we use comprehensive molecular characterization by whole genome/exome, transcriptome and methylome analysis in 70 CUP patients to reveal substantial mutational heterogeneity with TP53, MUC16, KRAS, LRP1B and CSMD3 being the most frequently mutated known cancer-related genes. The most common fusion partner is FGFR2, the most common focal homozygous deletion affects CDKN2A. 56/70 (80%) patients receive genomics-based treatment recommendations which are applied in 20/56 (36%) cases. Transcriptome and methylome data provide evidence for the underlying entity in 62/70 (89%) cases. Germline analysis reveals five (likely) pathogenic mutations in five patients. Recommended off-label therapies translate into a mean PFS ratio of 3.6 with a median PFS1 of 2.9 months (17 patients) and a median PFS2 of 7.8 months (20 patients). Our data emphasize the clinical value of molecular analysis and underline the need for innovative, mechanism-based clinical trials.


Assuntos
Neoplasias Primárias Desconhecidas , Epigenômica , Genômica , Homozigoto , Humanos , Mutação , Neoplasias Primárias Desconhecidas/tratamento farmacológico , Neoplasias Primárias Desconhecidas/genética , Deleção de Sequência
12.
Eur J Cancer ; 172: 107-118, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35763870

RESUMO

BACKGROUND: The multi-receptor tyrosine kinase inhibitor pazopanib is approved for the treatment of advanced soft-tissue sarcoma and has also shown activity in other sarcoma subtypes. However, its clinical efficacy is highly variable, and no reliable predictors exist to select patients who are likely to benefit from this drug. PATIENTS AND METHODS: We analysed the molecular profiles and clinical outcomes of patients with pazopanib-treated sarcoma enrolled in a prospective observational study by the German Cancer Consortium, DKTK MASTER, that employs whole-genome/exome sequencing and transcriptome sequencing to inform the care of young adults with advanced cancer across histology and patients with rare cancers. RESULTS: Among 109 patients with available whole-genome/exome sequencing data, there was no correlation between clinical parameters, specific genetic alterations or mutational signatures and clinical outcome. In contrast, the analysis of a subcohort of 62 patients who underwent molecular analysis before pazopanib treatment and had transcriptome sequencing data available showed that mRNA levels of NTRK3 (hazard ratio [HR] = 0.53, p = 0.021), IGF1R (HR = 1.82, p = 0.027) and KDR (HR = 0.50, p = 0.011) were independently associated with progression-free survival (PFS). Based on the expression of these receptor tyrosine kinase genes, i.e. the features NTRK3-high, IGF1R-low and KDR-high, we developed a pazopanib efficacy predictor that stratified patients into three groups with significantly different PFS (p < 0.0001). Application of the pazopanib efficacy predictor to an independent cohort of patients with pazopanib-treated sarcoma from DKTK MASTER (n = 43) confirmed its potential to separate patient groups with significantly different PFS (p = 0.02), whereas no such association was observed in patients with sarcoma from DKTK MASTER (n = 97) or The Cancer Genome Atlas sarcoma cohort (n = 256) who were not treated with pazopanib. CONCLUSION: A score based on the combined expression of NTRK3, IGF1R and KDR allows the identification of patients with sarcoma and with good, intermediate and poor outcome following pazopanib therapy and warrants prospective investigation as a predictive tool to optimise the use of this drug in the clinic.


Assuntos
Sarcoma , Neoplasias de Tecidos Moles , Expressão Gênica , Humanos , Indazóis/uso terapêutico , Estudos Prospectivos , Pirimidinas , Sarcoma/tratamento farmacológico , Sarcoma/genética , Neoplasias de Tecidos Moles/tratamento farmacológico , Sulfonamidas , Adulto Jovem
13.
Artigo em Inglês | MEDLINE | ID: mdl-34036222

RESUMO

PURPOSE: CATCH (Comprehensive Assessment of clinical feaTures and biomarkers to identify patients with advanced or metastatic breast Cancer for marker driven trials in Humans) is a prospective precision oncology program that uses genomics and transcriptomics to guide therapeutic decisions in the clinical management of metastatic breast cancer. Herein, we report our single-center experience and results on the basis of the first 200 enrolled patients of an ongoing trial. METHODS: From June 2017 to March 2019, 200 patients who had either primary metastatic or progressive disease, with any number of previous treatment lines and at least one metastatic site accessible to biopsy, were enrolled. DNA and RNA from tumor tissue and corresponding blood-derived nontumor DNA were profiled using whole-genome and transcriptome sequencing. Identified actionable alterations were brought into clinical context in a multidisciplinary molecular tumor board (MTB) with the aim of prioritizing personalized treatment recommendations. RESULTS: Among the first 200 enrolled patients, 128 (64%) were discussed in the MTB, of which 64 (50%) were subsequently treated according to MTB recommendation. Of 53 evaluable patients, 21 (40%) achieved either stable disease (n = 13, 25%) or partial response (n = 8, 15%). Furthermore, 16 (30%) of those patients showed improvement in progression-free survival of at least 30% while on MTB-recommended treatment compared with the progression-free survival of the previous treatment line. CONCLUSION: The initial phase of this study demonstrates that precision oncology on the basis of whole-genome and RNA sequencing is feasible when applied in the clinical management of patients with metastatic breast cancer and provides clinical benefit to a substantial proportion of patients.


Assuntos
Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Medicina de Precisão , Adulto , Idoso , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Feminino , Genoma , Humanos , Pessoa de Meia-Idade , Metástase Neoplásica , Estudos Prospectivos , Transcriptoma
14.
Cancer Discov ; 11(11): 2780-2795, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34112699

RESUMO

The clinical relevance of comprehensive molecular analysis in rare cancers is not established. We analyzed the molecular profiles and clinical outcomes of 1,310 patients (rare cancers, 75.5%) enrolled in a prospective observational study by the German Cancer Consortium that applies whole-genome/exome and RNA sequencing to inform the care of adults with incurable cancers. On the basis of 472 single and six composite biomarkers, a cross-institutional molecular tumor board provided evidence-based management recommendations, including diagnostic reevaluation, genetic counseling, and experimental treatment, in 88% of cases. Recommended therapies were administered in 362 of 1,138 patients (31.8%) and resulted in significantly improved overall response and disease control rates (23.9% and 55.3%) compared with previous therapies, translating into a progression-free survival ratio >1.3 in 35.7% of patients. These data demonstrate the benefit of molecular stratification in rare cancers and represent a resource that may promote clinical trial access and drug approvals in this underserved patient population. SIGNIFICANCE: Rare cancers are difficult to treat; in particular, molecular pathogenesis-oriented medical therapies are often lacking. This study shows that whole-genome/exome and RNA sequencing enables molecularly informed treatments that lead to clinical benefit in a substantial proportion of patients with advanced rare cancers and paves the way for future clinical trials.See related commentary by Eggermont et al., p. 2677.This article is highlighted in the In This Issue feature, p. 2659.


Assuntos
Neoplasias , Transcriptoma , Adulto , Perfilação da Expressão Gênica , Genômica , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Sequenciamento do Exoma
15.
Mol Metab ; 24: 98-107, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30956117

RESUMO

OBJECTIVE: Genome wide association studies (GWAS) for type 2 diabetes (T2D) have identified genetic loci that often localise in non-coding regions of the genome, suggesting gene regulation effects. We combined genetic and transcriptomic analysis from human islets obtained from brain-dead organ donors or surgical patients to detect expression quantitative trait loci (eQTLs) and shed light into the regulatory mechanisms of these genes. METHODS: Pancreatic islets were isolated either by laser capture microdissection (LCM) from surgical specimens of 103 metabolically phenotyped pancreatectomized patients (PPP) or by collagenase digestion of pancreas from 100 brain-dead organ donors (OD). Genotyping (> 8.7 million single nucleotide polymorphisms) and expression (> 47,000 transcripts and splice variants) analyses were combined to generate cis-eQTLs. RESULTS: After applying genome-wide false discovery rate significance thresholds, we identified 1,173 and 1,021 eQTLs in samples of OD and PPP, respectively. Among the strongest eQTLs shared between OD and PPP were CHURC1 (OD p-value=1.71 × 10-24; PPP p-value = 3.64 × 10-24) and PSPH (OD p-value = 3.92 × 10-26; PPP p-value = 3.64 × 10-24). We identified eQTLs in linkage-disequilibrium with GWAS loci T2D and associated traits, including TTLL6, MLX and KIF9 loci, which do not implicate the nearest gene. We found in the PPP datasets 11 eQTL genes, which were differentially expressed in T2D and two genes (CYP4V2 and TSEN2) associated with HbA1c but none in the OD samples. CONCLUSIONS: eQTL analysis of LCM islets from PPP led us to identify novel genes which had not been previously linked to islet biology and T2D. The understanding gained from eQTL approaches, especially using surgical samples of living patients, provides a more accurate 3-dimensional representation than those from genetic studies alone.


Assuntos
Diabetes Mellitus Tipo 2/genética , Ilhotas Pancreáticas/metabolismo , Locos de Características Quantitativas , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Família 4 do Citocromo P450/genética , Diabetes Mellitus Tipo 2/patologia , Humanos , Cinesinas/genética , Microdissecção e Captura a Laser , Proteínas de Membrana/genética , Peptídeo Sintases/genética , Polimorfismo de Nucleotídeo Único
16.
Nat Commun ; 10(1): 1635, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30967556

RESUMO

Chordomas are rare bone tumors with few therapeutic options. Here we show, using whole-exome and genome sequencing within a precision oncology program, that advanced chordomas (n = 11) may be characterized by genomic patterns indicative of defective homologous recombination (HR) DNA repair and alterations affecting HR-related genes, including, for example, deletions and pathogenic germline variants of BRCA2, NBN, and CHEK2. A mutational signature associated with HR deficiency was significantly enriched in 72.7% of samples and co-occurred with genomic instability. The poly(ADP-ribose) polymerase (PARP) inhibitor olaparib, which is preferentially toxic to HR-incompetent cells, led to prolonged clinical benefit in a patient with refractory chordoma, and whole-genome analysis at progression revealed a PARP1 p.T910A mutation predicted to disrupt the autoinhibitory PARP1 helical domain. These findings uncover a therapeutic opportunity in chordoma that warrants further exploration, and provide insight into the mechanisms underlying PARP inhibitor resistance.


Assuntos
Cordoma/tratamento farmacológico , Ftalazinas/uso terapêutico , Piperazinas/uso terapêutico , Poli(ADP-Ribose) Polimerase-1/genética , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Reparo de DNA por Recombinação/genética , Adulto , Idoso , Cordoma/genética , Cordoma/patologia , Mapeamento Cromossômico , Quebras de DNA de Cadeia Dupla , Análise Mutacional de DNA , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Instabilidade Genômica , Humanos , Masculino , Pessoa de Meia-Idade , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Medicina de Precisão/métodos , Domínios Proteicos/genética , Resultado do Tratamento , Sequenciamento do Exoma
17.
JMIR Cancer ; 4(1): e10160, 2018 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-29752255

RESUMO

This article describes the DataBox project which offers a perspective of a new health data management solution in Germany. DataBox was initially conceptualized as a repository of individual lung cancer patient data (structured and unstructured). The patient is the owner of the data and is able to share his or her data with different stakeholders. Data is transferred, displayed, and stored online, but not archived. In the long run, the project aims at replacing the conventional method of paper- and storage-device-based handling of data for all patients in Germany, leading to better organization and availability of data which reduces duplicate diagnostic procedures, treatment errors, and enables the training as well as usage of artificial intelligence algorithms on large datasets.

18.
J Clin Endocrinol Metab ; 103(12): 4373-4383, 2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30202879

RESUMO

Context: Reduced ß-cell mass, impaired islet function, and dedifferentiation are considered causal to development of hyperglycemia and type 2 diabetes. In human cohort studies, changes of islet cell-specific expression patterns have been associated with diabetes but not directly with in vivo insulin secretion. Objective: This study investigates alterations of islet gene expression and corresponding gene variants in the context of in vivo glycemic traits from the same patients. Methods: Fasting blood was collected before surgery, and pancreatic tissue was frozen after resection from 18 patients undergoing pancreatectomy. Islet tissue was isolated by laser capture microdissection. Islet transcriptome was analyzed using microarray and quantitative RT-PCR. Proteins were examined by immunohistochemistry and western blotting. The association of gene variants with insulin secretion was investigated with oral glucose tolerance test (OGTT)-derived insulin secretion measured in a large cohort of subjects at increased risk of type 2 diabetes and with hyperglycemic clamp in a subset. Results: Differential gene expression between islets from normoglycemic and hyperglycemic patients was prominent for the glycolytic enzyme ALDOB and the obesity-associated gene FAIM2. The mRNA levels of both genes correlated negatively with insulin secretion and positively with HbA1c. Islets of hyperglycemic patients displayed increased ALDOB immunoreactivity in insulin-positive cells, whereas α- and δ-cells were negative. Exposure of isolated islets to hyperglycemia augmented ALDOB expression. The minor allele of the ALDOB variant rs550915 associated with significantly higher levels of C-peptide and insulin during OGTT and hyperglycemic clamp, respectively. Conclusion: Our analyses suggest that increased ALDOB expression in human islets is associated with lower insulin secretion.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Hiperglicemia/metabolismo , Secreção de Insulina/fisiologia , Ilhotas Pancreáticas/metabolismo , Glicemia , Células Cultivadas , Estudos Transversais , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/genética , Frutose-Bifosfato Aldolase/genética , Perfilação da Expressão Gênica , Técnica Clamp de Glucose , Teste de Tolerância a Glucose , Hemoglobinas Glicadas/análise , Voluntários Saudáveis , Humanos , Hiperglicemia/sangue , Hiperglicemia/genética , Insulina/sangue , Microdissecção e Captura a Laser , Pancreatectomia , Neoplasias Pancreáticas/cirurgia , Polimorfismo de Nucleotídeo Único , Cultura Primária de Células
19.
JCO Precis Oncol ; 2: 1-13, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-35135162

RESUMO

PURPOSE: Rapidly evolving genomics technologies, in particular comprehensive next-generation sequencing (NGS), have led to exponential growth in the understanding of cancer biology, shifting oncology toward personalized treatment strategies. However, comprehensive NGS approaches, such as whole-exome sequencing, have limitations that are related to the technology itself as well as to the input source. Hence, clinical implementation of comprehensive NGS in a quality-controlled diagnostic workflow requires both the standardization of sequencing procedures and continuous validation of sequencing results by orthogonal methods in an ongoing program to enable the determination of key test parameters and continuous improvement of NGS and bioinformatics pipelines. PATIENTS AND METHODS: We present validation data on 220 patients who were enrolled between 2013 and 2016 in a multi-institutional, genomics-guided precision oncology program (Molecularly Aided Stratification for Tumor Eradication Research) of the National Center for Tumor Diseases Heidelberg and the German Cancer Consortium. RESULTS: More than 90% of clinically actionable genomic alterations identified by combined whole-exome sequencing and transcriptome sequencing were successfully validated, with varying frequencies of discordant results across different types of alterations (fusions, 3.7%; single-nucleotide variants, 2.6%; amplifications, 1.1%; overexpression, 0.9%; deletions, 0.6%). The implementation of new computational methods for NGS data analysis led to a substantial improvement of gene fusion calling over time. CONCLUSION: Collectively, these data demonstrate the value of a rigorous validation program that partners with comprehensive NGS to successfully implement and continuously improve cancer precision medicine in a clinical setting.

20.
Cancer Discov ; 8(9): 1087-1095, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29802158

RESUMO

We used whole-genome and transcriptome sequencing to identify clinically actionable genomic alterations in young adults with pancreatic ductal adenocarcinoma (PDAC). Molecular characterization of 17 patients with PDAC enrolled in a precision oncology program revealed gene fusions amenable to pharmacologic inhibition by small-molecule tyrosine kinase inhibitors in all patients with KRAS wild-type (KRASWT) tumors (4 of 17). These alterations included recurrent NRG1 rearrangements predicted to drive PDAC development through aberrant ERBB receptor-mediated signaling, and pharmacologic ERBB inhibition resulted in clinical improvement and remission of liver metastases in 2 patients with NRG1-rearranged tumors that had proved resistant to standard treatment. Our findings demonstrate that systematic screening of KRASWT tumors for oncogenic fusion genes will substantially improve the therapeutic prospects for a sizeable fraction of patients with PDAC.Significance: Advanced PDAC is a malignancy with few treatment options that lacks molecular mechanism-based therapies. Our study uncovers recurrent gene rearrangements such as NRG1 fusions as disease-driving events in KRASwt tumors, thereby providing novel insights into oncogenic signaling and new therapeutic options in this entity. Cancer Discov; 8(9); 1087-95. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 1047.


Assuntos
Carcinoma Ductal Pancreático/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Neuregulina-1/genética , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas p21(ras)/genética , Adulto , Animais , Carcinoma Ductal Pancreático/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundário , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas de Fusão Oncogênica/genética , Neoplasias Pancreáticas/genética , Medicina de Precisão , Inibidores de Proteínas Quinases/farmacologia , Bibliotecas de Moléculas Pequenas/administração & dosagem , Bibliotecas de Moléculas Pequenas/farmacologia , Translocação Genética , Resultado do Tratamento , Sequenciamento Completo do Genoma/métodos , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto Jovem
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