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AIMS AND BACKGROUND: Whole-genome sequencing (WGS) is increasingly applied in clinical practice and expected to replace standard-of-care (SoC) genetic diagnostics in hematological malignancies. This study aims to assess and compare the fully burdened cost ('micro-costing') per patient for Swedish laboratories using WGS and SoC, respectively, in pediatric and adult patients with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). METHODS: The resource use and cost details associated with SoC, e.g. chromosome banding analysis, fluorescent in situ hybridization, and targeted sequencing analysis, were collected via activity-based costing methods from four diagnostic laboratories. For WGS, corresponding data was collected from two of the centers. A simulation-based scenario model was developed for analyzing the WGS cost based on different annual sample throughput to evaluate economy of scale. RESULTS: The average SoC total cost per patient was 2,465 for pediatric AML and 2,201 for pediatric ALL, while in adults, the corresponding cost was 2,458 for AML and 1,207 for ALL. The average WGS cost (90x tumor/30x normal; sequenced on the Illumina NovaSeq 6000 platform) was estimated to 3,472 based on an annual throughput of 2,500 analyses, however, with an annual volume of 7,500 analyses the average cost would decrease by 23% to 2,671. CONCLUSION: In summary, WGS is currently more costly than SoC, however the cost can be reduced by utilizing laboratories with higher throughput and by the expected decline in cost of reagents. Our data provides guidance to decision-makers for the resource allocation needed when implementing WGS in diagnostics of hematological malignancies.
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Pruebas Genéticas , Leucemia Mieloide Aguda , Leucemia-Linfoma Linfoblástico de Células Precursoras , Secuenciación Completa del Genoma , Humanos , Suecia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/diagnóstico , Secuenciación Completa del Genoma/economía , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Pruebas Genéticas/economía , Pruebas Genéticas/métodos , Adulto , Niño , Masculino , Femenino , Costos y Análisis de CostoRESUMEN
Gene panel sequencing has become a common diagnostic tool for detecting somatically acquired mutations in myeloid neoplasms. However, many panels have restricted content, provide insufficient sensitivity levels, or lack clinically validated workflows. We here describe the development and validation of the Genomic Medicine Sweden myeloid gene panel (GMS-MGP), a capture-based 191 gene panel including mandatory genes in contemporary guidelines as well as emerging candidates. The GMS-MGP displayed uniform coverage across all targets, including recognized difficult GC-rich areas. The validation of 117 previously described somatic variants showed a 100% concordance with a limit-of-detection of a 0.5% variant allele frequency (VAF), achieved by utilizing error correction and filtering against a panel-of-normals. A national interlaboratory comparison investigating 56 somatic variants demonstrated highly concordant results in both detection rate and reported VAFs. In addition, prospective analysis of 323 patients analyzed with the GMS-MGP as part of standard-of-care identified clinically significant genes as well as recurrent mutations in less well-studied genes. In conclusion, the GMS-MGP workflow supports sensitive detection of all clinically relevant genes, facilitates novel findings, and is, based on the capture-based design, easy to update once new guidelines become available. The GMS-MGP provides an important step toward nationally harmonized precision diagnostics of myeloid malignancies.
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Medicina de Precisión , Humanos , Medicina de Precisión/métodos , Mutación , Suecia , Pruebas Genéticas/métodos , Pruebas Genéticas/normas , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/diagnóstico , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Frecuencia de los GenesRESUMEN
PURPOSE: Clinical relapse is the major threat for patients with myelodysplastic syndrome (MDS) undergoing hematopoietic stem-cell transplantation (HSCT). Early detection of measurable residual disease (MRD) would enable preemptive treatment and potentially reduced relapse risk. METHODS: Patients with MDS planned for HSCT were enrolled in a prospective, observational study evaluating the association between MRD and clinical outcome. We collected bone marrow (BM) and peripheral blood samples until relapse, death, or end of study 24 months after HSCT. Patient-specific mutations were identified with targeted next-generation sequencing (NGS) panel and traced using droplet digital polymerase chain reaction (ddPCR). RESULTS: Of 266 included patients, estimated relapse-free survival (RFS) and overall survival (OS) rates 3 years after HSCT were 59% and 64%, respectively. MRD results were available for 221 patients. Relapse was preceded by positive BM MRD in 42/44 relapses with complete MRD data, by a median of 71 (23-283) days. Of 137 patients in continuous complete remission, 93 were consistently MRD-negative, 39 reverted from MRD+ to MRD-, and five were MRD+ at last sampling. Estimated 1 year-RFS after first positive MRD was 49%, 39%, and 30%, using cutoff levels of 0.1%, 0.3%, and 0.5%, respectively. In a multivariate Cox model, MRD (hazard ratio [HR], 7.99), WHO subgroup AML (HR, 4.87), TP53 multi-hit (HR, 2.38), NRAS (HR, 3.55), and acute GVHD grade III-IV (HR, 4.13) were associated with shorter RFS. MRD+ was also independently associated with shorter OS (HR, 2.65). In a subgroup analysis of 100 MRD+ patients, presence of chronic GVHD was associated with longer RFS (HR, 0.32). CONCLUSION: Assessment of individualized MRD using NGS + ddPCR is feasible and can be used for early detection of relapse. Positive MRD is associated with shorter RFS and OS (ClinicalTrials.gov identifier: NCT02872662).
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Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Leucemia Mieloide Aguda , Síndromes Mielodisplásicos , Humanos , Enfermedad Injerto contra Huésped/etiología , Trasplante de Células Madre Hematopoyéticas/métodos , Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicos/terapia , Recurrencia Local de Neoplasia/genética , Neoplasia Residual/genética , Pronóstico , Estudios Prospectivos , RecurrenciaRESUMEN
Patients with chronic lymphocytic leukemia (CLL) progressing on ibrutinib constitute an unmet need. Though Bruton tyrosine kinase (BTK) and PLCG2 mutations are associated with ibrutinib resistance, their frequency and relevance to progression are not fully understood. In this multicenter retrospective observational study, we analyzed 98 patients with CLL on ibrutinib (49 relapsing after an initial response and 49 still responding after ≥1 year of continuous treatment) using a next-generation sequencing (NGS) panel (1% sensitivity) comprising 13 CLL-relevant genes including BTK and PLCG2. BTK hotspot mutations were validated by droplet digital polymerase chain reaction (ddPCR) (0.1% sensitivity). By integrating NGS and ddPCR results, 32 of 49 relapsing cases (65%) carried at least 1 hotspot BTK and/or PLCG2 mutation(s); in 6 of 32, BTK mutations were only detected by ddPCR (variant allele frequency [VAF] 0.1% to 1.2%). BTK/PLCG2 mutations were also identified in 6 of 49 responding patients (12%; 5/6 VAF <10%), of whom 2 progressed later. Among the relapsing patients, the BTK-mutated (BTKmut) group was enriched for EGR2 mutations, whereas BTK-wildtype (BTKwt) cases more frequently displayed BIRC3 and NFKBIE mutations. Using an extended capture-based panel, only BRAF and IKZF3 mutations showed a predominance in relapsing cases, who were enriched for del(8p) (n = 11; 3 BTKwt). Finally, no difference in TP53 mutation burden was observed between BTKmut and BTKwt relapsing cases, and ibrutinib treatment did not favor selection of TP53-aberrant clones. In conclusion, we show that BTK/PLCG2 mutations were absent in a substantial fraction (35%) of a real-world cohort failing ibrutinib, and propose additional mechanisms contributing to resistance.
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Leucemia Linfocítica Crónica de Células B , Humanos , Agammaglobulinemia Tirosina Quinasa/genética , Leucemia Linfocítica Crónica de Células B/diagnóstico , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Resistencia a Antineoplásicos/genética , Piperidinas , RecurrenciaRESUMEN
The clinical significance of small TP53 clones detected with next generation sequencing (NGS) in chronic lymphocytic leukemia is an issue of active debate. According to the official guidelines, treatment decisions should be guided only by variants with variant allele frequency (VAF) ≥10%. We present data on 325 consecutive patients with chronic lymphocytic leukemia analyzed with NGS. In total 47 pathogenic/likely pathogenic (P/LP), TP53 variants were detected in 26 patients (8%). Eleven of these (23%) were in the 5% to 10% VAF range and reported according to our institutional policy. All TP53 variants in the 5% to 10% VAF range were confirmed (100% concordance) with a second NGS panel. Our results where further validated with the performance of Sanger sequencing and digital droplet PCR (ddPCR). In 12 patients with available fluorescence in situ hybridization data and TP53 mutations within 5% to 10% VAF, deletion of chromosome 17p (del(17p)) was detectable in only 1 patient. We propose a robust diagnostic algorithm, which allows the safe detection and reporting of TP53 variants with VAF down to 5% in the clinical setting. Our study provides evidence that NGS is equally potent to detect variants with VAF 5% to 10% compared to those with VAF 10% to 15%, highlighting the urgent need for harmonization of NGS methodologies across diagnostic laboratories.
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Rare tumor-specific mutations in patient samples serve as excellent markers to monitor the course of malignant disease and responses to therapy in clinical routine, and improved assay techniques are needed for broad adoption. We describe herein a highly sensitive and selective molecule amplification technology - superRCA assays - for rapid and highly specific detection of DNA sequence variants present at very low frequencies in DNA samples. Using a standard flow cytometer we demonstrate precise, ultra-sensitive detection of single-nucleotide mutant sequences from malignant cells against up to a 100,000-fold excess of DNA from normal cells in either bone marrow or peripheral blood, to follow the course of patients treated for acute myeloid leukemia (AML). We also demonstrate that sequence variants located in a high-GC region may be sensitively detected, and we illustrate the potential of the technology for early detection of disease recurrence as a basis for prompt change of therapy.
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Leucemia Mieloide Aguda , Secuencia de Bases , Médula Ósea , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , MutaciónRESUMEN
Germline pathogenic variants in RUNX1 are associated with familial platelet disorder with predisposition to myeloid malignancies (FPD/MM) with intragenic deletions in RUNX1 accounting for almost 7% of all reported variants. We present two new pedigrees with FPD/MM carrying two different germline RUNX1 intragenic deletions. The aforementioned deletions encompass exons 1-2 and 9-10 respectively, with the exon 9-10 deletion being previously unreported. RNA sequencing of patients carrying the exon 9-10 deletion revealed a fusion with LINC00160 resulting in a change in the 3' sequence of RUNX1. Expression analysis of the transcript isoform demonstrated altered RUNX1a/b/c ratios in carriers from both families compared to controls. Our data provide evidence on the impact of intragenic RUNX1 deletions on transcript isoform expression and highlight the importance of routinely performing copy number variant analysis in patients with suspected MM with germline predisposition.
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Trastornos de las Plaquetas Sanguíneas , Leucemia Mieloide Aguda , Trastornos de las Plaquetas Sanguíneas/complicaciones , Trastornos de las Plaquetas Sanguíneas/diagnóstico , Trastornos de las Plaquetas Sanguíneas/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Exones , Células Germinativas/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Isoformas de Proteínas/genéticaRESUMEN
Clonal haematopoiesis of indeterminate potential (CHIP) may predispose for the development of therapy-related myeloid neoplasms (t-MN). Using target next-generation sequencing (t-NGS) panels and digital droplet polymerase chain reactions (ddPCR), we studied the myeloid gene mutation profiles of patients with chronic lymphocytic leukaemia (CLL) who developed a t-MN after treatment with chemo-(immuno)therapy. Using NGS, we detected a total of 30 pathogenic/likely pathogenic (P/LP) variants in 10 of 13 patients with a t-MN (77%, median number of variants for patient: 2, range 0-6). The prevalence of CHIP was then backtracked in paired samples taken at CLL diagnosis in eight of these patients. Six of them carried at least one CHIP-variant at the time of t-MN (median: 2, range: 1-5), and the same variants were present in the CLL sample in five cases. CHIP variants were present in 34 of 285 patients from a population-based CLL cohort, which translates into a significantly higher prevalence of CHIP in patients with a CLL who developed a t-MN, compared to the population-based cohort (5/8, 62.5% vs. 34/285, 12%, p = 0.0001). Our data show that CHIP may be considered as a novel parameter affecting treatment algorithms in patients with CLL, and highlight the potential of using chemo-free therapies in CHIP-positive cases.
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Leucemia Linfocítica Crónica de Células B , Neoplasias Primarias Secundarias , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Hematopoyesis Clonal/genética , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Mutación , Neoplasias Primarias Secundarias/etiología , Neoplasias Primarias Secundarias/genética , Factores de RiesgoRESUMEN
Next-generation sequencing (NGS) has transitioned from research to clinical routine, yet the comparability of different technologies for mutation profiling remains an open question. We performed a European multicenter (n=6) evaluation of three amplicon-based NGS assays targeting 11 genes recurrently mutated in chronic lymphocytic leukemia. Each assay was assessed by two centers using 48 pre-characterized chronic lymphocytic leukemia samples; libraries were sequenced on the Illumina MiSeq instrument and bioinformatics analyses were centralized. Across all centers the median percentage of target reads ≥100x ranged from 94.2- 99.8%. In order to rule out assay-specific technical variability, we first assessed variant calling at the individual assay level i.e., pairwise analysis of variants detected amongst partner centers. After filtering for variants present in the paired normal sample and removal of PCR/sequencing artefacts, the panels achieved 96.2% (Multiplicom), 97.7% (TruSeq) and 90% (HaloPlex) concordance at a variant allele frequency (VAF) >0.5%. Reproducibility was assessed by looking at the inter-laboratory variation in detecting mutations and 107 of 115 (93% concordance) mutations were detected by all six centers, while the remaining eight variants (7%) were undetected by a single center. Notably, 6 of 8 of these variants concerned minor subclonal mutations (VAF <5%). We sought to investigate low-frequency mutations further by using a high-sensitivity assay containing unique molecular identifiers, which confirmed the presence of several minor subclonal mutations. Thus, while amplicon-based approaches can be adopted for somatic mutation detection with VAF >5%, after rigorous validation, the use of unique molecular identifiers may be necessary to reach a higher sensitivity and ensure consistent and accurate detection of low-frequency variants.
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Leucemia Linfocítica Crónica de Células B , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Leucemia Linfocítica Crónica de Células B/diagnóstico , Leucemia Linfocítica Crónica de Células B/genética , Mutación , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: The histone 3 lysine 4 (H3K4) monomethylase KMT2C is mutated across several cancer types; however, the effects of mutations on epigenome organization, gene expression, and cell growth are not clear. A frequently recurring mutation in colorectal cancer (CRC) with microsatellite instability is a single nucleotide deletion within the exon 38 poly-A(9) repeat (c.8390delA) which results in frameshift preceding the functional carboxy-terminal SET domain. To study effects of KMT2C expression in CRC cells, we restored one allele to wild type KMT2C in the two CRC cell lines RKO and HCT116, which both are homozygous c.8390delA mutant. RESULTS: Gene editing resulted in increased KMT2C expression, increased H3K4me1 levels, altered gene expression profiles, and subtle negative effects on cell growth, where higher dependence and stronger effects of KMT2C expression were observed in RKO compared to HCT116 cells. Surprisingly, we found that the two RKO and HCT116 CRC cell lines have distinct baseline H3K4me1 epigenomic profiles. In RKO cells, a flatter genome-wide H3K4me1 profile was associated with more increased H3K4me1 deposition at enhancers, reduced cell growth, and more differential gene expression relative to HCT116 cells when KMT2C was restored. Profiling of H3K4me1 did not indicate a highly specific regulation of gene expression as KMT2C-induced H3K4me1 deposition was found globally and not at a specific enhancer sub-set in the engineered cells. Although we observed variation in differentially regulated gene sets between cell lines and individual clones, differentially expressed genes in both cell lines included genes linked to known cancer signaling pathways, estrogen response, hypoxia response, and aspects of immune system regulation. CONCLUSIONS: Here, KMT2C restoration reduced CRC cell growth and reinforced genome-wide H3K4me1 deposition at enhancers; however, the effects varied depending upon the H3K4me1 status of KMT2C deficient cells. Results indicate that KMT2C inactivation may promote colorectal cancer development through transcriptional dysregulation in several pathways with known cancer relevance.
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Neoplasias Colorrectales/genética , Proteínas de Unión al ADN/genética , Histonas/metabolismo , Variantes Farmacogenómicas/genética , Alelos , Proliferación Celular/genética , Metilación de ADN/genética , Epigénesis Genética/genética , Exones/genética , Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/genética , Estudio de Asociación del Genoma Completo/métodos , Células HCT116 , Humanos , Inestabilidad de Microsatélites , Mutación , Transducción de SeñalRESUMEN
The tumor cells in diffuse large B-cell lymphomas (DLBCL) are considered to originate from germinal center derived B-cells (GCB) or activated B-cells (ABC). Gene expression profiling (GEP) is preferably used to determine the cell of origin (COO). However, GEP is not widely applied in clinical practice and consequently, several algorithms based on immunohistochemistry (IHC) have been developed. Our aim was to evaluate the concordance of COO assignment between the Lymph2Cx GEP assay and the IHC-based Hans algorithm, to decide which model is the best survival predictor. Both GEP and IHC were performed in 359 homogenously treated Swedish and Danish DLBCL patients, in a retrospective multicenter cohort. The overall concordance between GEP and IHC algorithm was 72%; GEP classified 85% of cases assigned as GCB by IHC, as GCB, while 58% classified as non-GCB by IHC, were categorized as ABC by GEP. There were significant survival differences (overall survival and progression-free survival) if cases were classified by GEP, whereas if cases were categorized by IHC only progression-free survival differed significantly. Importantly, patients assigned as non-GCB/ABC both by IHC and GEP had the worst prognosis, which was also significant in multivariate analyses. Double expression of MYC and BCL2 was more common in ABC cases and was associated with a dismal outcome. In conclusion, to determine COO both by IHC and GEP is the strongest outcome predictor to identify DLBCL patients with the worst outcome.
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Linfocitos B/citología , Inmunohistoquímica/métodos , Linfoma de Células B Grandes Difuso/mortalidad , Pronóstico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Algoritmos , Dinamarca , Perfilación de la Expresión Génica , Centro Germinal/citología , Humanos , Activación de Linfocitos , Linfoma de Células B Grandes Difuso/diagnóstico , Persona de Mediana Edad , Estudios Retrospectivos , Análisis de Supervivencia , Suecia , Adulto JovenAsunto(s)
Antimetabolitos Antineoplásicos/uso terapéutico , Azacitidina/uso terapéutico , Linfadenopatía Inmunoblástica/tratamiento farmacológico , Linfoma de Células T/tratamiento farmacológico , Síndromes Mielodisplásicos/tratamiento farmacológico , Anciano de 80 o más Años , Antimetabolitos Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Azacitidina/farmacología , Médula Ósea/efectos de los fármacos , Médula Ósea/patología , Ciclofosfamida/farmacología , Ciclofosfamida/uso terapéutico , ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN/efectos de los fármacos , ADN Metiltransferasa 3A , Proteínas de Unión al ADN/genética , Dioxigenasas , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Resistencia a Antineoplásicos/genética , Femenino , Proteínas de Homeodominio/genética , Humanos , Linfadenopatía Inmunoblástica/diagnóstico , Linfadenopatía Inmunoblástica/genética , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/patología , Linfoma de Células T/diagnóstico , Linfoma de Células T/genética , Mutación , Síndromes Mielodisplásicos/genética , Prednisona/farmacología , Prednisona/uso terapéutico , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Factores de Transcripción/genética , Resultado del Tratamiento , Vincristina/farmacología , Vincristina/uso terapéuticoRESUMEN
Despite the recent discovery of recurrent driver mutations in chronic lymphocytic leukemia, the genetic factors involved in disease onset remain largely unknown. To address this issue, we performed whole-genome sequencing in 11 individuals with monoclonal B- cell lymphocytosis, both of the low-count and high-count subtypes, and 5 patients with ultra-stable chronic lymphocytic leukemia (>10 years without progression from initial diagnosis). All three entities were indistinguishable at the genomic level exhibiting low genomic complexity and similar types of somatic mutations. Exonic mutations were not frequently identified in putative chronic lymphocytic leukemia driver genes in all settings, including low-count monoclonal B-cell lymphocytosis. To corroborate these findings, we also performed deep sequencing in 11 known frequently mutated genes in an extended cohort of 28 monoclonal B-cell lymphocytosis/chronic lymphocytic leukemia cases. Interestingly, shared mutations were detected between clonal B cells and paired polymorphonuclear cells, strengthening the notion that at least a fraction of somatic mutations may occur before disease onset, likely at the hematopoietic stem cell level. Finally, we identified previously unreported non-coding variants targeting pathways relevant to B-cell and chronic lymphocytic leukemia development, likely associated with the acquisition of the characteristic neoplastic phenotype typical of both monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia.
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Linfocitos B/patología , Biomarcadores de Tumor/genética , Genómica/métodos , Leucemia Linfocítica Crónica de Células B/genética , Linfocitosis/genética , Mutación , Estudios de Cohortes , Progresión de la Enfermedad , Humanos , Leucemia Linfocítica Crónica de Células B/patología , Linfocitosis/patología , Fenotipo , Pronóstico , Secuenciación Completa del GenomaRESUMEN
The chromatin modifier PRDM2/RIZ1 is inactivated by mutation in several forms of cancer and is a putative tumor suppressor gene. Frameshift mutations in the C-terminal region of PRDM2, affecting (A)8 or (A)9 repeats within exon 8, are found in one third of colorectal cancers with microsatellite instability, but the contribution of these mutations to colorectal tumorigenesis is unknown. To model somatic mutations in microsatellite unstable tumors, we devised a general approach to perform genome editing while stabilizing the mutated nucleotide repeat. We then engineered isogenic cell systems where the PRDM2 c.4467delA mutation in human HCT116 colorectal cancer cells was corrected to wild-type by genome editing. Restored PRDM2 increased global histone 3 lysine 9 dimethylation and reduced migration, anchorage-independent growth and tumor growth in vivo. Gene set enrichment analysis revealed regulation of several hallmark cancer pathways, particularly of epithelial-to-mesenchymal transition (EMT), with VIM being the most significantly regulated gene. These observations provide direct evidence that PRDM2 c.4467delA is a driver mutation in colorectal cancer and confirms PRDM2 as a cancer gene, pointing to regulation of EMT as a central aspect of its tumor suppressive action.
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BACKGROUND: The disco-interacting protein 2 homolog C (DIP2C) gene is an uncharacterized gene found mutated in a subset of breast and lung cancers. To understand the role of DIP2C in tumour development we studied the gene in human cancer cells. METHODS: We engineered human DIP2C knockout cells by genome editing in cancer cells. The growth properties of the engineered cells were characterised and transcriptome and methylation analyses were carried out to identify pathways deregulated by inactivation of DIP2C. Effects on cell death pathways and epithelial-mesenchymal transition traits were studied based on the results from expression profiling. RESULTS: Knockout of DIP2C in RKO cells resulted in cell enlargement and growth retardation. Expression profiling revealed 780 genes for which the expression level was affected by the loss of DIP2C, including the tumour-suppressor encoding CDKN2A gene, the epithelial-mesenchymal transition (EMT) regulator-encoding ZEB1, and CD44 and CD24 that encode breast cancer stem cell markers. Analysis of DNA methylation showed more than 30,000 sites affected by differential methylation, the majority of which were hypomethylated following loss of DIP2C. Changes in DNA methylation at promoter regions were strongly correlated to changes in gene expression, and genes involved with EMT and cell death were enriched among the differentially regulated genes. The DIP2C knockout cells had higher wound closing capacity and showed an increase in the proportion of cells positive for cellular senescence markers. CONCLUSIONS: Loss of DIP2C triggers substantial DNA methylation and gene expression changes, cellular senescence and epithelial-mesenchymal transition in cancer cells.
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Proteínas Portadoras/genética , Neoplasias del Colon/genética , Metilación de ADN/genética , Transición Epitelial-Mesenquimal/genética , Proteínas Nucleares/genética , Proteínas Portadoras/antagonistas & inhibidores , Línea Celular Tumoral , Senescencia Celular/genética , Neoplasias del Colon/patología , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Nucleares/antagonistas & inhibidores , Transcriptoma/genéticaRESUMEN
BACKGROUND: The thiosemicarbazone CD 02750 (VLX50) was recently reported as a hit compound in a phenotype-based drug screen in primary cultures of patient tumor cells. We synthesized a copper complex of VLX50, denoted VLX60, and characterized its antitumor and mechanistic properties. MATERIALS AND METHODS: The cytotoxic effects and mechanistic properties of VLX60 were investigated in monolayer cultures of multiple human cell lines, in tumor cells from patients, in a 3-D spheroid cell culture system and in vivo and were compared with those of VLX50. RESULTS: VLX60 showed ≥ 3-fold higher cytotoxic activity than VLX50 in 2-D cultures and, in contrast to VLX50, retained its activity in the presence of additional iron. VLX60 was effective against non-proliferative spheroids and against tumor xenografts in vivo in a murine model. In contrast to VLX50, gene expression analysis demonstrated that genes associated with oxidative stress were considerably enriched in cells exposed to VLX60 as was induction of reactive oxygen. VLX60 compromised the ubiquitin-proteasome system and was more active in BRAF mutated versus BRAF wild-type colon cancer cells. CONCLUSIONS: The cytotoxic effects of the copper thiosemicarbazone VLX60 differ from those of VLX50 and shows interesting features as a potential antitumor drug, notably against BRAF mutated colorectal cancer.
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Antineoplásicos/farmacología , Cobre , Piridinas/farmacología , Tiosemicarbazonas/farmacología , Animales , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cobre/química , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Estrés Oxidativo/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Piridinas/química , Tiosemicarbazonas/química , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We recently reported a truncating deletion in the NFKBIE gene, which encodes IκBε, a negative feedback regulator of NF-κB, in clinically aggressive chronic lymphocytic leukemia (CLL). Because preliminary data indicate enrichment of NFKBIE aberrations in other lymphoid malignancies, we screened a large patient cohort (n = 1460) diagnosed with different lymphoid neoplasms. While NFKBIE deletions were infrequent in follicular lymphoma, splenic marginal zone lymphoma, and T-cell acute lymphoblastic leukemia (<2%), slightly higher frequencies were seen in diffuse large B-cell lymphoma, mantle cell lymphoma, and primary central nervous system lymphoma (3% to 4%). In contrast, a remarkably high frequency of NFKBIE aberrations (46/203 cases [22.7%]) was observed in primary mediastinal B-cell lymphoma (PMBL) and Hodgkin lymphoma (3/11 cases [27.3%]). NFKBIE-deleted PMBL patients were more often therapy refractory (P = .022) and displayed inferior outcome compared with wild-type patients (5-year survival, 59% vs 78%; P = .034); however, they appeared to benefit from radiotherapy (P =022) and rituximab-containing regimens (P = .074). NFKBIE aberrations remained an independent factor in multivariate analysis (P = .003) and when restricting the analysis to immunochemotherapy-treated patients (P = .008). Whole-exome sequencing and gene expression profiling verified the importance of NF-κB deregulation in PMBL. In summary, we identify NFKBIE aberrations as a common genetic event across B-cell malignancies and highlight NFKBIE deletions as a novel poor-prognostic marker in PMBL.