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AIM: To explore the experiences of registered nurses providing care to adult patients affected by encephalitis, from admission into hospital through to discharge. STUDY DESIGN: A qualitative phenomenological methodology was used. Sample and setting: Eight registered nurses in a city centre teaching hospital. METHODS: Data collection took place using in-depth, semi-structured interviews. Data were analysed and themes identified using framework analysis. FINDINGS: Three key findings were identified: nurses felt that they lacked knowledge of encephalitis, lacked time to give these patients the care they needed, and they lacked access to rehabilitation for patients with encephalitis. CONCLUSION: This study provides the first evidence on nurses' experiences of providing care to patients affected by encephalitis. It has shown that they often lack the knowledge and time to give adequate support to patients. They also lack access to rehabilitation for these patients.
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Encefalitis , Enfermeras y Enfermeros , Personal de Enfermería en Hospital , Adulto , Hospitales , Humanos , Conocimiento , Atención al Paciente , Investigación CualitativaRESUMEN
BACKGROUND: The importance of chimerism status in the very early period after hematopoietic stem cell transplantation is unclear. We determined PBMC and T-cell donor chimerism 50 days after transplantation and related this to disease relapse and overall survival. METHODS: 144 sequential patients underwent transplantation of which 90 had AML/MDS and 54 had lymphoma. 'Full donor chimerism' was defined as ≥99% donor cells and three patient groups were defined: 40% with full donor chimerism (FC) in both PBMC and T-cells; 25% with mixed chimerism (MC) within both compartments and 35% with 'split' chimerism (SC) characterised by full donor chimerism within PBMC and mixed chimerism within T-cells. RESULTS: In patients with myeloid disease a pattern of mixed chimerism (MC) was associated with a one year relapse rate of 45% and a five year overall survival of 40% compared to values of 8% and 75%, and 17% and 60%, for those with SC or FC respectively. The pattern of chimerism had no impact on clinical outcome for lymphoma. CONCLUSION: The pattern of lineage-specific chimerism at 50 days after transplantation is highly predictive of clinical outcome for patients with myeloid malignancy and may help to guide subsequent clinical management.
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Trasplante de Células Madre Hematopoyéticas , Leucemia Mieloide Aguda , Linfoma , Síndromes Mielodisplásicos , Linfocitos T/metabolismo , Quimera por Trasplante/sangre , Adulto , Anciano , Aloinjertos , Supervivencia sin Enfermedad , Femenino , Humanos , Leucemia Mieloide Aguda/sangre , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/terapia , Linfoma/sangre , Linfoma/mortalidad , Linfoma/terapia , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/sangre , Síndromes Mielodisplásicos/mortalidad , Síndromes Mielodisplásicos/terapia , Tasa de SupervivenciaRESUMEN
OBJECTIVES: To develop a focused panel of somatic mutations (SMs) present in the majority of urothelial bladder cancers (UBCs), to investigate the diagnostic and prognostic utility of this panel, and to compare the identification of SMs in urinary cell-pellet (cp)DNA and cell-free (cf)DNA as part of the development of a non-invasive clinical assay. PATIENTS AND METHODS: A panel of SMs was validated by targeted deep-sequencing of tumour DNA from 956 patients with UBC. In addition, amplicon and capture-based targeted sequencing measured mutant allele frequencies (MAFs) of SMs in 314 urine cpDNAs and 153 urine cfDNAs. The association of SMs with grade, stage, and clinical outcomes was investigated by univariate and multivariate Cox models. Concordance between SMs detected in tumour tissue and cpDNA and cfDNA was assessed. RESULTS: The panel comprised SMs in 23 genes: TERT (promoter), FGFR3, PIK3CA, TP53, ERCC2, RHOB, ERBB2, HRAS, RXRA, ELF3, CDKN1A, KRAS, KDM6A, AKT1, FBXW7, ERBB3, SF3B1, CTNNB1, BRAF, C3orf70, CREBBP, CDKN2A, and NRAS; 93.5-98.3% of UBCs of all grades and stages harboured ≥1 SM (mean: 2.5 SMs/tumour). RAS mutations were associated with better overall survival (P = 0.04). Mutations in RXRA, RHOB and TERT (promoter) were associated with shorter time to recurrence (P < 0.05). MAFs in urinary cfDNA and cpDNA were highly correlated; using a capture-based approach, >94% of tumour SMs were detected in both cpDNA and cfDNA. CONCLUSIONS: SMs are reliably detected in urinary cpDNA and cfDNA. The technical capability to identify very low MAFs is essential to reliably detect UBC, regardless of the use of cpDNA or cfDNA. This 23-gene panel shows promise for the non-invasive diagnosis and risk stratification of UBC.
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ADN de Neoplasias/orina , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mutación/genética , Neoplasias de la Vejiga Urinaria/diagnóstico , Neoplasias de la Vejiga Urinaria/genética , Adulto , Anciano , Anciano de 80 o más Años , Bases de Datos Genéticas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Medición de Riesgo , Análisis de Secuencia de ADNRESUMEN
The live attenuated Japanese encephalitis (JE) vaccine SA14-14-2 has been used in Nepal for catch-up campaigns and is now included in the routine immunisation schedule. Previous studies have shown good vaccine efficacy after one dose in districts with a high incidence of JE. The first well-documented dengue outbreak occurred in Nepal in 2006 with ongoing cases now thought to be secondary to migration from India. Previous infection with dengue virus (DENV) partially protects against JE and might also influence serum neutralising antibody titres against JEV. This study aimed to determine whether serum anti-JEV neutralisation titres are: 1. maintained over time since vaccination, 2. vary with historic local JE incidence, and 3. are associated with DENV neutralising antibody levels. We conducted a cross-sectional study in three districts of Nepal: Banke, Rupandehi and Udayapur. Udayapur district had been vaccinated against JE most recently (2009), but had been the focus of only one campaign, compared with two in Banke and three in Rupandehi. Participants answered a short questionnaire and serum was assayed for anti-JEV and anti-DENV IgM and IgG (by ELISA) and 50% plaque reduction neutralisation titres (PRNT50) against JEV and DENV serotypes 1-4. A titre of ≥1:10 was considered seropositive to the respective virus. JEV neutralising antibody seroprevalence (PRNT50 ≥ 1:10) was 81% in Banke and Rupandehi, but only 41% in Udayapur, despite this district being vaccinated more recently. Sensitivity of ELISA for both anti-JEV and anti-DENV antibodies was low compared with PRNT50. DENV neutralising antibody correlated with the JEV PRNT50 ≥1:10, though the effect was modest. IgM (indicating recent infection) against both viruses was detected in a small number of participants. We also show that DENV IgM is present in Nepali subjects who have not travelled to India, suggesting that DENV may have become established in Nepal. We therefore propose that further JE vaccine campaigns should be considered in Udayapur district, and similar areas that have had fewer vaccination campaigns.
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Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Virus de la Encefalitis Japonesa (Especie)/inmunología , Encefalitis Japonesa/epidemiología , Encefalitis Japonesa/prevención & control , Programas de Inmunización , Vacunas contra la Encefalitis Japonesa/administración & dosificación , Adulto , Anciano , Anciano de 80 o más Años , Estudios Transversales , Virus del Dengue/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Masculino , Persona de Mediana Edad , Nepal/epidemiología , Pruebas de Neutralización , Estudios Seroepidemiológicos , Encuestas y Cuestionarios , Ensayo de Placa Viral , Adulto JovenRESUMEN
Transplantation is an effective treatment of many clinical disorders, but the mechanisms that regulate immunological tolerance are uncertain and remain central to improving patient outcome. Hemopoietic stem cell transplantation (SCT) often establishes "mixed chimerism" in which immune cells from both the donor and patient coexist in vivo in a setting of immunological tolerance. We studied immune function in 69 patients within 2 months following SCT; 37 were fully donor and 32 displayed mixed chimerism. The proportion of T regulatory (Treg) cells was increased during mixed chimerism and comprised equal numbers of donor and host-derived regulatory cells. This was associated with a tolerogenic PD-L1+ profile on dendritic cells. Importantly, effector T cells from patients with mixed chimerism exhibited reduced cytotoxicity against host target cells in vitro, but this was restored following depletion of CD4+ Treg cells. These data show that Treg cells play a major role in sustaining immunological tolerance during mixed chimerism. These insights should help to guide novel interventions to improve clinical transplantation.
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Quimerismo , Trasplante de Células Madre Hematopoyéticas , Linfocitos T Reguladores/inmunología , Antígeno B7-H1/metabolismo , Células Dendríticas/metabolismo , Reacción Injerto-Huésped/inmunología , Humanos , Tolerancia Inmunológica/inmunología , Linfocitos T Reguladores/citología , Donantes de Tejidos , Tolerancia al Trasplante/inmunologíaRESUMEN
INTRODUCTION: Phase I of the Cancer Research UK Stratified Medicine Programme (SMP1) was designed to roll out molecular pathology testing nationwide at the point of cancer diagnosis, as well as facilitate an infrastructure where surplus cancer tissue could be used for research. It offered a non-trial setting to examine common UK cancer genetics in a real-world context. METHODS: A total of 26 sites in England, Wales and Scotland, recruited samples from 7814 patients for genetic examination between 2011 and 2013. Tumour types involved were breast, colorectal, lung, prostate, ovarian cancer and malignant melanoma. Centralised molecular testing of surplus material from resections or biopsies of primary/metastatic tissue was performed, with samples examined for 3-5 genetic alterations deemed to be of key interest in site-specific cancers by the National Cancer Research Institute Clinical Study groups. RESULTS: 10 754 patients (98% of those approached) consented to participate, from which 7814 tumour samples were genetically analysed. In total, 53% had at least one genetic aberration detected. From 1885 patients with lung cancer, KRAS mutation was noted to be highly prevalent in adenocarcinoma (37%). In breast cancer (1873 patients), there was a striking contrast in TP53 mutation incidence between patients with ductal cancer (27.3%) and lobular cancer (3.4%). Vast inter-tumour heterogeneity of colorectal cancer (1550 patients) was observed, including myriad double and triple combinations of genetic aberrations. Significant losses of important clinical information included smoking status in lung cancer and loss of distinction between low-grade and high-grade serous ovarian cancers. CONCLUSION: Nationwide molecular pathology testing in a non-trial setting is feasible. The experience with SMP1 has been used to inform ongoing CRUK flagship programmes such as the CRUK National Lung MATRIX trial and TRACERx.
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Trastornos Linfoproliferativos/tratamiento farmacológico , Ftalazinas/uso terapéutico , Piperazinas/uso terapéutico , Anciano , Proteínas de la Ataxia Telangiectasia Mutada/genética , Daño del ADN/genética , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/mortalidad , Leucemia Prolinfocítica de Células T/tratamiento farmacológico , Leucemia Prolinfocítica de Células T/mortalidad , Linfoma de Células del Manto/tratamiento farmacológico , Linfoma de Células del Manto/mortalidad , Trastornos Linfoproliferativos/mortalidad , Masculino , Dosis Máxima Tolerada , Persona de Mediana Edad , Ftalazinas/administración & dosificación , Ftalazinas/efectos adversos , Piperazinas/administración & dosificación , Piperazinas/efectos adversos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/efectos adversos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Recurrencia , Análisis de Supervivencia , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Subclonal heterogeneity and clonal selection influences disease progression in chronic lymphocytic leukemia (CLL). It is therefore important that therapeutic decisions are made based on an understanding of the CLL clonal architecture and its dynamics in individual patients. Identification of cytogenetic abnormalities by FISH remains the cornerstone of contemporary clinical practice and provides a simple means for prognostic stratification. Here, we demonstrate that multiplexed-FISH can enhance recognition of CLL subclonal repertoire and its dynamics during disease progression, both in patients and CLL patient-derived xenografts (PDX). We applied a combination of patient-specific FISH probes to 24 CLL cases before treatment and at relapse, and determined putative ancestral relationships between subpopulations with different cytogenetic features. We subsequently established 7 CLL PDX models in NOD/Shi-SCID/IL-2Rγctm1sug/Jic (NOG) mice. Application of multiplexed-FISH to these models demonstrated that all of the identified cytogenetic subpopulations had leukemia propagating activity and that changes in their representation during disease progression could be spontaneous, accelerated by treatment or treatment-induced. We conclude that multiplexed-FISH in combination with PDX models have the potential to distinguish between spontaneous and treatment-induced clonal selection, and therefore provide a valuable tool for the pre-clinical evaluation of novel therapies.
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Aberraciones Cromosómicas , Evolución Clonal/genética , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Animales , Terapia Combinada , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Xenoinjertos , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Leucemia Linfocítica Crónica de Células B/mortalidad , Leucemia Linfocítica Crónica de Células B/terapia , Masculino , Ratones , Pronóstico , Análisis de la Célula Individual , Resultado del TratamientoRESUMEN
Although technically possible, few clinical laboratories across the world have implemented non-invasive prenatal diagnosis (NIPD) for selected single-gene disorders, mostly owing to the elevated costs incurred. Having previously proven that NIPD for X-linked disorders can be feasibly implemented in clinical practice, we have now developed a test for the NIPD of an autosomal-recessive disorder, spinal muscular atrophy (SMA). Cell-free DNA was extracted from maternal blood and prepared for massively parallel sequencing on an Illumina MiSeq by targeted capture enrichment of single-nucleotide polymorphisms across a 6 Mb genomic window on chromosome 5 containing the SMN1 gene. Maternal, paternal and proband DNA samples were also tested for haplotyping purposes. Sequencing data was analysed by relative haplotype dosage (RHDO). Six pregnant SMA carriers and 10 healthy pregnant donors were recruited through the NIPSIGEN study. Inheritance of the maternally and paternally derived alleles of the affected SMN1 gene was determined in the foetus by RHDO analysis for autosomal-recessive disorders. DNA from the proband (for SMA carriers) or an invasively obtained foetal sample (for healthy pregnant donors) was used to identify the maternal and paternal reference haplotypes associated with the affected SMN1 gene. Results for all patients correlated with known outcomes and showed a testing specificity and sensitivity of 100%. On top of showing high accuracy and reliability throughout the stages of validation, our novel test for NIPD of SMA is also affordable and viable for implementation into clinical service.
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Pruebas Genéticas/métodos , Haplotipos , Atrofia Muscular Espinal/diagnóstico , Diagnóstico Prenatal/métodos , Estudios de Casos y Controles , Femenino , Pruebas Genéticas/normas , Heterocigoto , Humanos , Masculino , Atrofia Muscular Espinal/genética , Linaje , Polimorfismo de Nucleótido Simple , Embarazo , Diagnóstico Prenatal/normas , Sensibilidad y Especificidad , Proteína 1 para la Supervivencia de la Neurona Motora/genéticaRESUMEN
Our understanding of the perturbation of normal cellular differentiation hierarchies to create tumor-propagating stem cell populations is incomplete. In human acute myeloid leukemia (AML), current models suggest transformation creates leukemic stem cell (LSC) populations arrested at a progenitor-like stage expressing cell surface CD34. We show that in â¼25% of AML, with a distinct genetic mutation pattern where >98% of cells are CD34(-), there are multiple, nonhierarchically arranged CD34(+) and CD34(-) LSC populations. Within CD34(-) and CD34(+) LSC-containing populations, LSC frequencies are similar; there are shared clonal structures and near-identical transcriptional signatures. CD34(-) LSCs have disordered global transcription profiles, but these profiles are enriched for transcriptional signatures of normal CD34(-) mature granulocyte-macrophage precursors, downstream of progenitors. But unlike mature precursors, LSCs express multiple normal stem cell transcriptional regulators previously implicated in LSC function. This suggests a new refined model of the relationship between LSCs and normal hemopoiesis in which the nature of genetic/epigenetic changes determines the disordered transcriptional program, resulting in LSC differentiation arrest at stages that are most like either progenitor or precursor stages of hemopoiesis.
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Antígenos CD34/genética , Células Progenitoras de Granulocitos y Macrófagos/metabolismo , Leucemia Mieloide Aguda , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/metabolismo , Animales , Antígenos CD34/metabolismo , Células Progenitoras de Granulocitos y Macrófagos/patología , Xenoinjertos , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas de Neoplasias/metabolismo , Trasplante de Neoplasias , Células Madre Neoplásicas/patologíaRESUMEN
BACKGROUND: Cytomegalovirus (CMV) is a highly prevalent herpesvirus, which maintains lifelong latency and places a significant burden on host immunity. Infection is associated with increased rates of vascular disease and overall mortality in the elderly and there is an urgent need for improved understanding of the viral-host balance during ageing. CMV is extremely difficult to detect in healthy donors, however, using droplet digital PCR of DNA from peripheral blood monocytes, we obtained an absolute quantification of viral load in 44 healthy donors across a range of ages. RESULTS: Viral DNA was detected in 24 % (9/37) of donors below the age of 70 but was found in all individuals above this age. Furthermore, the mean CMV load was only 8.6 copies per 10,000 monocytes until approximately 70 years of age when it increased by almost 30 fold to 249 copies in older individuals (p < 0.0001). CMV was found within classical CD14+ monocytes and was not detectable within the CD14-CD16+ subset. The titre of CMV-specific IgG increased inexorably with age indicating that loss of humoral immunity is not a determinant of the increased viral load. In contrast, although cellular immunity to the structural late protein pp65 increased with age, the T cell response to the immediate early protein IE1 decreased in older donors. CONCLUSION: These data reveal that effective control of CMV is impaired during healthy ageing, most probably due to loss of cellular control of early viral reactivation. This information will be of value in guiding efforts to reduce CMV-associated health complications in the elderly.
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Urothelial bladder cancers (UBCs) have heterogeneous clinical characteristics that are mirrored in their diverse genomic profiles. Genomic profiling of UBCs has the potential to benefit routine clinical practice by providing prognostic utility above and beyond conventional clinicopathological factors, and allowing for prediction and surveillance of treatment responses. Urinary DNAs representative of the tumour genome provide a promising resource as a liquid biopsy for non-invasive genomic profiling of UBCs. We compared the genomic profiles of urinary cellular DNA and cell-free DNA (cfDNA) from the urine with matched diagnostic formalin-fixed paraffin-embedded tumour DNAs for 23 well-characterised UBC patients. Our data show urinary DNAs to be highly representative of patient tumours, allowing for detection of recurrent clinically actionable genomic aberrations. Furthermore, a greater aberrant load (indicative of tumour genome) was observed in cfDNA over cellular DNA (P<0.001), resulting in a higher analytical sensitivity for detection of clinically actionable genomic aberrations (P<0.04) when using cfDNA. Thus, cfDNA extracted from the urine of UBC patients has a higher tumour genome burden and allows greater detection of key genomic biomarkers (90%) than cellular DNA from urine (61%) and provides a promising resource for robust whole-genome tumour profiling of UBC with potential to influence clinical decisions without invasive patient interventions.
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Biomarcadores de Tumor/orina , Carcinoma/genética , ADN de Neoplasias/orina , Genoma Humano , Neoplasias de la Vejiga Urinaria/genética , Biomarcadores de Tumor/genética , Carcinoma/patología , Carcinoma/orina , Aberraciones Cromosómicas , ADN de Neoplasias/genética , Humanos , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/orina , Urotelio/metabolismo , Urotelio/patologíaRESUMEN
OBJECTIVE: Development of an accurate and affordable test for the non-invasive prenatal diagnosis of Duchenne and Becker muscular dystrophies (DMD/BMD) to implement in clinical practice. METHOD: Cell-free DNA was extracted from maternal blood and prepared for massively parallel sequencing on an Illumina MiSeq by targeted capture enrichment of single nucleotide polymorphisms (SNPs) across the dystrophin gene on chromosome X. Sequencing data were analysed by relative haplotype dosage. RESULTS: Seven healthy pregnant donors and two pregnant DMD carriers all bearing a male fetus were recruited through the non-invasive prenatal diagnosis for single gene disorders study. Non-invasive prenatal diagnosis testing was conducted by relative haplotype dosage analysis for X-linked disorders where the genomic DNA from the chorionic villus sampling (for healthy pregnant donors) or from the proband (for pregnant DMD carriers) was used to identify the reference haplotype. Results for all patients showed a test accuracy of 100%, when the calculated fetal fraction was >4% and correlated with known outcomes. A recombination event was also detected in a DMD patient. CONCLUSION: Our new test for NIPD of DMD/BMD has been shown to be accurate and reliable during initial stages of validation. It is also feasible for implementation into clinical service.
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Distrofina/genética , Pruebas Genéticas/métodos , Haplotipos , Pruebas de Detección del Suero Materno/métodos , Distrofia Muscular de Duchenne/diagnóstico , Polimorfismo de Nucleótido Simple , Estudios de Casos y Controles , Sistema Libre de Células , ADN/sangre , Femenino , Marcadores Genéticos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Distrofia Muscular de Duchenne/genética , EmbarazoRESUMEN
Disease relapse is the major cause of treatment failure after allogeneic stem cell transplantation (allo-SCT) in acute myeloid leukemia (AML). To identify AML-associated genes prognostic of AML relapse post-allo-SCT, we resequenced 35 genes in 113 adults at diagnosis, 49 of whom relapsed. Two hundred sixty-two mutations were detected in 102/113 (90%) patients. An increased risk of relapse was observed in patients with mutations in WT1 (P = .018), DNMT3A (P = .045), FLT3 ITD (P = .071), and TP53 (P = .06), whereas mutations in IDH1 were associated with a reduced risk of disease relapse (P = .018). In 29 patients, we additionally compared mutational profiles in bone marrow at diagnosis and relapse to study changes in clonal structure at relapse. In 13/29 patients, mutational profiles altered at relapse. In 9 patients, mutations present at relapse were not detected at diagnosis. In 15 patients, additional available pre-allo-SCT samples demonstrated that mutations identified posttransplant but not at diagnosis were detectable immediately prior to transplant in 2 of 15 patients. Taken together, these observations, if confirmed in larger studies, have the potential to inform the design of novel strategies to reduce posttransplant relapse highlighting the potential importance of post-allo-SCT interventions with a broad antitumor specificity in contrast to targeted therapies based on mutational profile at diagnosis.
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BACKGROUND: Adoption of new technology in both basic research and clinical settings requires rigorous validation of analytical performance. The OncoScan® FFPE Assay is a multiplexing tool that offers genome-wide copy number and loss of heterozygosity detection, as well as identification of frequently tested somatic mutations. METHODS: In this study, 162 formalin fixed paraffin embedded samples, representing six different tumour types, were profiled in triplicate across three independent laboratories. OncoScan® formalin fixed paraffin embedded assay data was then analysed for reproducibility of genome-wide copy number, loss of heterozygosity and somatic mutations. Where available, somatic mutation data was compared to data from orthogonal technologies (pyro/sanger sequencing). RESULTS: Cross site comparisons of genome-wide copy number and loss of heterozygosity profiles showed greater than 95% average agreement between sites. Somatic mutations pre-validated by orthogonal technologies showed greater than 90% agreement with OncoScan® somatic mutation calls and somatic mutation concordance between sites averaged 97%. CONCLUSIONS: Reproducibility of whole-genome copy number, loss of heterozygosity and somatic mutation data using the OncoScan® assay has been demonstrated with comparatively low DNA inputs from a range of highly degraded formalin fixed paraffin embedded samples. In addition, our data shows examples of clinically-relevant aberrations that demonstrate the potential utility of the OncoScan® assay as a robust clinical tool for guiding tumour therapy.
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Técnicas de Laboratorio Clínico/normas , Perfilación de la Expresión Génica/métodos , Genoma Humano , Neoplasias/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Fijación del Tejido/métodos , Análisis Mutacional de ADN , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Pérdida de Heterocigocidad , Masculino , Mutación , Neoplasias/metabolismo , Adhesión en Parafina , Control de Calidad , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Análisis de Secuencia de ADNRESUMEN
Changes in gene dosage are a major driver of cancer, known to be caused by a finite, but increasingly well annotated, repertoire of mutational mechanisms. This can potentially generate correlated copy-number alterations across hundreds of linked genes, as exemplified by the 2% of childhood acute lymphoblastic leukaemia (ALL) with recurrent amplification of megabase regions of chromosome 21 (iAMP21). We used genomic, cytogenetic and transcriptional analysis, coupled with novel bioinformatic approaches, to reconstruct the evolution of iAMP21 ALL. Here we show that individuals born with the rare constitutional Robertsonian translocation between chromosomes 15 and 21, rob(15;21)(q10;q10)c, have approximately 2,700-fold increased risk of developing iAMP21 ALL compared to the general population. In such cases, amplification is initiated by a chromothripsis event involving both sister chromatids of the Robertsonian chromosome, a novel mechanism for cancer predisposition. In sporadic iAMP21, breakage-fusion-bridge cycles are typically the initiating event, often followed by chromothripsis. In both sporadic and rob(15;21)c-associated iAMP21, the final stages frequently involve duplications of the entire abnormal chromosome. The end-product is a derivative of chromosome 21 or the rob(15;21)c chromosome with gene dosage optimized for leukaemic potential, showing constrained copy-number levels over multiple linked genes. Thus, dicentric chromosomes may be an important precipitant of chromothripsis, as we show rob(15;21)c to be constitutionally dicentric and breakage-fusion-bridge cycles generate dicentric chromosomes somatically. Furthermore, our data illustrate that several cancer-specific mutational processes, applied sequentially, can coordinate to fashion copy-number profiles over large genomic scales, incrementally refining the fitness benefits of aggregated gene dosage changes.
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Aberraciones Cromosómicas , Cromosomas Humanos Par 21/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Cromátides/genética , Rotura Cromosómica , Cromosomas Humanos Par 15/genética , Variaciones en el Número de Copia de ADN/genética , Humanos , Recombinación Genética/genética , Translocación Genética/genéticaAsunto(s)
Lepra/rehabilitación , Pobreza/prevención & control , Derechos Humanos , Humanos , MianmarRESUMEN
BACKGROUND: ZAP70 gene expression is associated with poor prognosis in B-cell lymphoproliferative disorders especially chronic lymphocytic leukaemia (CLL) but its role in adult B-ALL has not been established. On diagnostic samples from 76 patients with adult ALL (65 with B-ALL and 11 with T-ALL) ZAP70 mRNA expression levels were studied by real time-quantitative PCR (RT-qPCR) analysis. FINDINGS: A broad distribution of ZAP70 expression was observed in ALL, ranging from 0.002 to 5.3 fold that of the ZAP70 positive Jurkat reference cell line. No association was observed between expression levels and the presence of specific cytogenetic abnormalities. Five cases, including one case of T-ALL, had ZAP70 expression above the level of the Jurkat reference cell line. CONCLUSIONS: Our results confirm the frequent expression of ZAP70 in adult ALL. Limited comparisons made did highlight poor-risk patients with high ZAP70 expression, but due to lack of clinical information on patient samples we were unable to directly assess the impact on disease prognosis. ZAP-70 may be an important laboratory assay in adult ALL and further studies are warranted to study a potential correlation with cytogenetic and other genetic markers.
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Strategies that augment a GVL effect without increasing the risk of GVHD are required to improve the outcome after allogeneic stem cell transplantation (SCT). Azacitidine (AZA) up-regulates the expression of tumor Ags on leukemic blasts in vitro and expands the numbers of immunomodulatory T regulatory cells (Tregs) in animal models. Reasoning that AZA might selectively augment a GVL effect, we studied the immunologic sequelae of AZA administration after allogeneic SCT. Twenty-seven patients who had undergone a reduced intensity allogeneic transplantation for acute myeloid leukemia were treated with monthly courses of AZA, and CD8(+) T-cell responses to candidate tumor Ags and circulating Tregs were measured. AZA after transplantation was well tolerated, and its administration was associated with a low incidence of GVHD. Administration of AZA increased the number of Tregs within the first 3 months after transplantation compared with a control population (P = .0127). AZA administration also induced a cytotoxic CD8(+) T-cell response to several tumor Ags, including melanoma-associated Ag 1, B melanoma antigen 1, and Wilm tumor Ag 1. These data support the further examination of AZA after transplantation as a mechanism of augmenting a GVL effect without a concomitant increase in GVHD.