RESUMEN
Dysfunctional homologous recombination DNA repair (HRR), frequently due to BRCA mutations, is a determinant of sensitivity to platinum chemotherapy and poly(ADP-ribose) polymerase inhibitors (PARPi). In cultures of ovarian cancer cells, we have previously shown that HRR function, based upon RAD51 foci quantification, correlated with growth inhibition ex vivo induced by rucaparib (a PARPi) and 12-month survival following platinum chemotherapy. The aim of this study was to determine the feasibility of measuring HRR dysfunction (HRD) in other tumours, in order to estimate the frequency and hence wider potential of PARPi. A total of 24 cultures were established from ascites sampled from 27 patients with colorectal, upper gastrointestinal, pancreatic, hepatobiliary, breast, mesothelioma, and non-epithelial ovarian cancers; 8 were HRD. Cell growth following continuous exposure to 10 µM of rucaparib was lower in HRD cultures compared to HRR-competent (HRC) cultures. Overall survival in the 10 patients who received platinum-based therapy was marginally higher in the 3 with HRD ascites (median overall survival of 17 months, range 10 to 90) compared to the 7 patients with HRC ascites (nine months, range 1 to 55). HRR functional assessment in primary cultures, from several tumour types, revealed that a third are HRD, justifying the further exploration of PARPi therapy in a broader range of tumours.
RESUMEN
New drugs are needed for the treatment of relapsed acute lymphoblastic leukemia and preclinical evaluation of the MEK inhibitor, selumetinib, has shown that this drug has excellent activity in those leukemias with RAS pathway mutations. The proapoptotic protein, BIM is pivotal in the induction of cell death by both selumetinib and glucocorticoids, suggesting the potential for synergy. Thus, combination indices for dexamethasone and selumetinib were determined in RAS pathway-mutated acute lymphoblastic leukemia primagraft cells in vitro and were indicative of strong synergism (combination index <0.2; n=5). Associated pharmacodynamic assays were consistent with the hypothesis that the drug combination enhanced BIM upregulation over that achieved by a single drug alone. Dosing of dexamethasone and selumetinib singly and in combination in mice engrafted with primary-derived RAS pathway-mutated leukemia cells resulted in a marked reduction in spleen size which was significantly greater with the drug combination. Assessment of the central nervous system leukemia burden showed a significant reduction in the drug-treated mice, with no detectable leukemia in those treated with the drug combination. These data suggest that a selumetinib-dexamethasone combination may be highly effective in RAS pathway-mutated acute lymphoblastic leukemia. An international phase I/II clinical trial of dexamethasone and selumetinib (Seludex trial) is underway in children with multiply relapsed/refractory disease.
Asunto(s)
Proteína 11 Similar a Bcl2/metabolismo , Bencimidazoles/administración & dosificación , Glucocorticoides/administración & dosificación , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas ras/genética , Adolescente , Animales , Niño , Preescolar , Análisis Mutacional de ADN , Dexametasona/administración & dosificación , Sinergismo Farmacológico , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mutación , Trasplante de Neoplasias , Regulación hacia ArribaRESUMEN
PARP inhibitors (PARPi) represent a major advance in the treatment of ovarian cancer associated with defects in homologous recombination DNA repair (HRR), primarily due to mutations in BRCA genes. Imatinib and PI3K inhibitors are reported to downregulate HRR and, in some cases, sensitise cells to PARPi. We investigated the ability of imatinib, and the PI3K inhibitors: NVP-BEZ235 and VS-5584, to downregulate HRR and sensitise paired ovarian cancer cells with mutant and reconstituted BRCA1 to the PARPi, olaparib and rucaparib. Olaparib and imatinib combinations were also measured in primary cultures of ovarian cancer. NVP-BEZ235 and imatinib reduced RAD51 levels and focus formation (an indication of HRR function), but VS-5584 did not. In colony-forming assays none of the inhibitors sensitised cells to PARPi cytotoxicity, in fact there was a mild protective effect. These conflicting data were resolved by the observation that the kinase inhibitors reduced the S-phase fraction, when HRR proteins are at their peak and cells are sensitive to PARPi cytotoxicity. In contrast, in primary cultures in 96-well plate assays, imatinib did increase olaparib-induced growth inhibition. However, in one primary culture that could be used in colony-formation cytotoxicity assays, imatinib protected from olaparib cytotoxicity. The kinase inhibitors protect from PARPi cytotoxicity by arresting cell growth, but this may be interpreted as synergy on the basis of 96-well cell growth assays. We urge caution before combining these drugs clinically.
Asunto(s)
Antineoplásicos/farmacología , Reparación del ADN/efectos de los fármacos , Neoplasias Ováricas/enzimología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Reparación del ADN/fisiología , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Mesilato de Imatinib/farmacología , Mesilato de Imatinib/uso terapéutico , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Ftalazinas/farmacología , Ftalazinas/uso terapéutico , Piperazinas/farmacología , Piperazinas/uso terapéutico , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
NUCOLL43 is a novel ovarian clear cell carcinoma (O-CCC) cell line that arose from a primary culture of a patient's malignant ascites. The cells grow reliably in cell culture with a doubling time of approx. 45 hours and form colonies at high efficiency. They have a very high degree of loss of heterozygosity (LOH) affecting approximately 85% of the genome, mostly copy neutral and almost identical to the original tumor. The cells express epithelial (pan-cytokeratin) and mesenchymal (vimentin) characteristics, CA125 and p16, like the original tumor. They also express ARID1A but not HNF-1ß and, like the original tumor, and are negative for p53 expression, with no evidence of p53 function. NUCOLL43 cells express all other DNA damage response proteins investigated and have functional homologous recombination DNA repair. They are insensitive to cisplatin, the PARP inhibitor rucaparib, and MDM2 inhibitors but are sensitive to camptothecin, paclitaxel, and NVP-BEZ235. The NUCOLL43 cell line represents a distinct subtype of O-CCC that is p53 and HNF-1ß null but expresses ARID1A. Its high degree of similarity with the original tumor genomically and proteomically, as well as the high level of LOH, make this an interesting cell line for O-CCC research. It has been deposited with Ximbio.
Asunto(s)
Adenocarcinoma de Células Claras/genética , Adenocarcinoma de Células Claras/patología , Resistencia a Antineoplásicos/genética , Estudio de Asociación del Genoma Completo , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Variantes Farmacogenómicas , Adenocarcinoma de Células Claras/diagnóstico por imagen , Adenocarcinoma de Células Claras/tratamiento farmacológico , Biopsia , Línea Celular Tumoral , Femenino , Genómica , Humanos , Repeticiones de Microsatélite , Persona de Mediana Edad , Neoplasias Ováricas/diagnóstico por imagen , Neoplasias Ováricas/tratamiento farmacológico , Fenotipo , Reparación del ADN por Recombinación , Tomografía Computarizada por Rayos XRESUMEN
Relapsed acute lymphoblastic leukemia is the most common cause of cancer-related mortality in young people and new therapeutic strategies are needed to improve outcome. Recent studies have shown that heterozygous inactivating mutations in the histone acetyl transferase, CREBBP, are particularly frequent in relapsed childhood acute lymphoblastic leukemia and associated with a hyperdiploid karyotype and KRAS mutations. To study the functional impact of CREBBP haploinsufficiency in acute lymphoblastic leukemia, RNA interference was used to knock down expression of CREBBP in acute lymphoblastic leukemia cell lines and various primagraft acute lymphoblastic leukemia cells. We demonstrate that attenuation of CREBBP results in reduced acetylation of histone 3 lysine 18, but has no significant impact on cAMP-dependent target gene expression. Impaired induction of glucocorticoid receptor targets was only seen in 1 of 4 CREBBP knockdown models, and there was no significant difference in glucocorticoid-induced apoptosis, sensitivity to other acute lymphoblastic leukemia chemotherapeutics or histone deacetylase inhibitors. Importantly, we show that CREBBP directly acetylates KRAS and that CREBBP knockdown enhances signaling of the RAS/RAF/MEK/ERK pathway in Ras pathway mutated acute lymphoblastic leukemia cells, which are still sensitive to MEK inhibitors. Thus, CREBBP mutations might assist in enhancing oncogenic RAS signaling in acute lymphoblastic leukemia but do not alter response to MEK inhibitors.
Asunto(s)
Proteína de Unión a CREB/deficiencia , Sistema de Señalización de MAP Quinasas , Mutación , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas ras/genética , Acetilación , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Línea Celular Tumoral , Modelos Animales de Enfermedad , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Histonas/metabolismo , Humanos , Ratones , Ratones Noqueados , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidad , Receptores de Glucocorticoides/metabolismo , Especificidad por Sustrato , Resultado del TratamientoRESUMEN
Somatic genetic abnormalities are initiators and drivers of disease and have proven clinical utility at initial diagnosis. However, the genetic landscape and its clinical utility at relapse are less well understood and have not been studied comprehensively. We analyzed cytogenetic data from 427 children with relapsed B-cell precursor ALL treated on the international trial, ALLR3. Also we screened 238 patients with a marrow relapse for selected copy number alterations (CNAs) and mutations. Cytogenetic risk groups were predictive of outcome postrelapse and survival rates at 5 years for patients with good, intermediate-, and high-risk cytogenetics were 68%, 47%, and 26%, respectively (P < .001). TP53 alterations and NR3C1/BTG1 deletions were associated with a higher risk of progression: hazard ratio 2.36 (95% confidence interval, 1.51-3.70, P < .001) and 2.15 (1.32-3.48, P = .002). NRAS mutations were associated with an increased risk of progression among standard-risk patients with high hyperdiploidy: 3.17 (1.15-8.71, P = .026). Patients classified clinically as standard and high risk had distinct genetic profiles. The outcome of clinical standard-risk patients with high-risk cytogenetics was equivalent to clinical high-risk patients. Screening patients at relapse for key genetic abnormalities will enable the integration of genetic and clinical risk factors to improve patient stratification and outcome. This study is registered at www.clinicaltrials.org as #ISCRTN45724312.
Asunto(s)
Predisposición Genética a la Enfermedad , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Adolescente , Niño , Preescolar , Aberraciones Cromosómicas , Estudios de Cohortes , Análisis Citogenético , Variaciones en el Número de Copia de ADN/genética , Demografía , Supervivencia sin Enfermedad , Femenino , Humanos , Lactante , Masculino , Mutación/genética , Pronóstico , Recurrencia , Factores de RiesgoRESUMEN
Glucocorticoid (GC) resistance is a continuing clinical problem in childhood acute lymphoblastic leukaemia (ALL) but the underlying mechanisms remain unclear. A proteomic approach was used to compare profiles of the B-lineage ALL GC-sensitive cell line, PreB 697, and its GC-resistant sub-line, R3F9, pre- and post-dexamethasone exposure. PAX5, a transcription factor critical to B-cell development was differentially regulated in the PreB 697 compared to the R3F9 cell line in response to GC. PAX5 basal protein expression was less in R3F9 compared to its GC-sensitive parent and confirmed to be lower in other GC-resistant sub-lines of Pre B 697 and was associated with a decreased expression of the PAX5 transcriptional target, CD19. Gene set enrichment analysis showed that increasing GC-resistance was associated with differentiation from preB-II to an immature B-lymphocyte stage. GC-resistant sub-lines were shown to have higher levels of phosphorylated JNK compared to the parent line and JNK inhibition caused re-sensitization to GC. Exploiting this maturation may be key to overcoming GC resistance and targeting signalling pathways linked to the maturation state, such as JNK, may be a novel approach.
Asunto(s)
Antineoplásicos/farmacología , Linfocitos B/efectos de los fármacos , Dexametasona/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , MAP Quinasa Quinasa 4/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas de Neoplasias/biosíntesis , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteómica/métodos , Apoptosis/efectos de los fármacos , Linfocitos B/patología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Antineoplásicos/fisiología , Exones/genética , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Reacción en Cadena de la Polimerasa Multiplex , Mutación , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Factor de Transcripción PAX5/genética , Factor de Transcripción PAX5/fisiología , Fosforilación/efectos de los fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/enzimología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa , Espectrometría de Masas en TándemRESUMEN
For most children who relapse with acute lymphoblastic leukemia (ALL), the prognosis is poor, and there is a need for novel therapies to improve outcome. We screened samples from children with B-lineage ALL entered into the ALL-REZ BFM 2002 clinical trial (www.clinicaltrials.gov, #NCT00114348) for somatic mutations activating the Ras pathway (KRAS, NRAS, FLT3, and PTPN11) and showed mutation to be highly prevalent (76 from 206). Clinically, they were associated with high-risk features including early relapse, central nervous system (CNS) involvement, and specifically for NRAS/KRAS mutations, chemoresistance. KRAS mutations were associated with a reduced overall survival. Mutation screening of the matched diagnostic samples found many to be wild type (WT); however, by using more sensitive allelic-specific assays, low-level mutated subpopulations were found in many cases, suggesting that they survived up-front therapy and subsequently emerged at relapse. Preclinical evaluation of the mitogen-activated protein kinase kinase 1/2 inhibitor selumetinib (AZD6244, ARRY-142886) showed significant differential sensitivity in Ras pathway-mutated ALL compared with WT cells both in vitro and in an orthotopic xenograft model engrafted with primary ALL; in the latter, reduced RAS-mutated CNS leukemia. Given these data, clinical evaluation of selumetinib may be warranted for Ras pathway-mutated relapsed ALL.
Asunto(s)
Bencimidazoles/uso terapéutico , Resistencia a Antineoplásicos/genética , Genes ras , Mutación , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Animales , Línea Celular Tumoral , Niño , Ensayos Clínicos como Asunto , Frecuencia de los Genes , Humanos , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Leucemia-Linfoma Linfoblástico de Células Precursoras/epidemiología , Recurrencia , Transducción de Señal/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Casitas B-lineage lymphoma (CBL) proteins are RING finger ubiquitin E3 ligases that attenuate the signaling of receptor tyrosine kinases and are mutated in a number of myeloid disorders. In this study, mutational screening of the linker-RING domains of CBL and CBLB was performed by denaturing high performance liquid chromatography in a cohort of diagnostic (n = 180) or relapse (n = 46) samples from children with acute lymphoblastic leukemia. Somatic mutations were identified in three children, giving an overall incidence of 1.7% and involved small deletions affecting the intron/exon boundaries of exon 8, leading to skipping of exon 8 and abolishing E3 ligase function. Mutated primary samples were associated with constitutive activation of the RAS pathway and sensitivity to MEK inhibitors was shown. Thus, mutation of CBL is an alternative route to activate the RAS pathway and may identify children who are candidates for MEK inhibitor clinical trials.
Asunto(s)
Mutación , Proteína Oncogénica v-cbl/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Transducción de Señal/genética , Adolescente , Secuencia de Bases , Niño , Preescolar , Cromatografía Liquida , Estudios de Cohortes , Análisis Mutacional de ADN , Exones , Femenino , Humanos , Intrones , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Masculino , Datos de Secuencia Molecular , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Dominios RING Finger , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Recurrencia , Reino UnidoRESUMEN
The mismatch repair (MMR) pathway is a post-replicative DNA repair process and MMR deficiency is a common feature of ALL cell lines. In this study we have investigated MMR deficiency in a large cohort of primary relapsed ALL (n=40) and investigated coding microsatellites (MS) of the lymphoid transcription factors, PAX5 and IKZF1 as downstream target genes. Only one patient showed MMR deficiency, as evidenced by microsatellite instability, which was acquired at relapse and was associated with reduced expression of both MLH1 and MSH2. Coding MS in candidate target genes including PAX5, IKZF1, BAX and TGFBRII were all wild type in this patient but the MMR-deficient cell line REH, was confirmed to have a coding MS in both PAX5 and TGFBRII. Whilst MMR deficiency is not highly prevalent in primary ALL, optimisation of the drug regimen to omit/replace thioguanines should be considered for children with MMR deficiency and/or reduced expression of key pathway components.
Asunto(s)
Reparación de la Incompatibilidad de ADN/genética , Factor de Transcripción Ikaros/genética , Factor de Transcripción PAX5/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas de Pez Cebra/genética , Adolescente , Western Blotting , Niño , Preescolar , Estudios de Cohortes , Metilación de ADN , Humanos , Factor de Transcripción Ikaros/metabolismo , Técnicas para Inmunoenzimas , Lactante , Inestabilidad de Microsatélites , Repeticiones de Microsatélite/genética , Mutación/genética , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/patología , Factor de Transcripción PAX5/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Proteínas de Pez Cebra/metabolismo , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Studies commonly report poor performance in psychotic patients compared with controls on tasks testing a range of cognitive functions, but, because current IQ is often not matched between these groups, it is difficult to determine whether this represents a generalized deficit or specific abnormalities. Fifty-three first-episode psychosis patients and 53 healthy controls, one-to-one matched for sex, age, and full-scale current IQ, were compared on Wechsler Adult Intelligence Scale (WAIS) subtests representing indices of perceptual organization, verbal comprehension, processing speed, and working memory as well as other tests of executive function and episodic memory. The groups showed an equivalent pattern of performance on all WAIS subtests except digit symbol processing speed, on which the patients were significantly worse. Patients were also worse on measures where performance correlated with digit symbol score, namely working and verbal memory tasks. Standardized residual scores for each subtest were calculated for each patient using the difference between their actual subtest score and a predicted subtest score based on their full-scale IQ and the performance of controls. Scaled scores and residual scores were examined for relationships with clinical measures. Digit symbol-scaled score was significantly correlated with concurrent negative syndrome score at baseline, and digit symbol residual score significantly predicted residual negative symptoms at 1-year follow-up. In summary, our comparison of patients and controls precisely matched for IQ revealed that processing speed was attenuated in recent-onset schizophrenia, contributed significantly to working and episodic memory deficits, and was a prognostic factor for poor outcome at 1 year.
Asunto(s)
Trastornos del Conocimiento/psicología , Función Ejecutiva , Inteligencia , Memoria a Corto Plazo , Trastornos Psicóticos/diagnóstico , Trastornos Psicóticos/psicología , Tiempo de Reacción , Esquizofrenia/diagnóstico , Psicología del Esquizofrénico , Escalas de Wechsler/estadística & datos numéricos , Adulto , Trastornos del Conocimiento/diagnóstico , Femenino , Estudios de Seguimiento , Humanos , Masculino , Psicometría , Valores de Referencia , Adulto JovenRESUMEN
Mismatch repair (MMR) deficiency is a common feature of acute lymphoblastic leukaemia (ALL) cell lines and in some cases is due to the mutations of hMLH1 which affect mRNA splicing. Therefore, we have analysed alternative splicing of hMLH1 in a cohort of children with ALL. We show that alternative splicing of hMLH1 is highly variable in normal and leukaemic cells and can occur by exon skipping or by the use of an alternative splice site, both serving to down-regulate the amount of full-length hMLH1 mRNA/protein produced. Aberrant splicing was found in one child with an aggressive leukaemia in which there was a predominant hMLH1Delta6 form and an associated loss of wild-type hMLH1 protein but this was not accompanied by microsatellite instability. Functional analysis of one of the most abundant spliced forms, hMLH1Delta9/10, was shown to have a significant dominant negative effect on the functionality of the MMR pathway but again was similarly expressed in ALL and normal cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Empalme Alternativo/genética , Proteínas Nucleares/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Western Blotting , Niño , Análisis Mutacional de ADN , Humanos , Repeticiones de Microsatélite , Homólogo 1 de la Proteína MutL , Isoformas de Proteínas , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
BACKGROUND: The intradimensional/extradimensional (IDED) task assesses different forms of learning from feedback. Limited evidence suggests that attentional set-shifting deteriorates over time in schizophrenia. We tested this hypothesis and examined the specificity of learning impairments identified by this task. METHOD: Two hundred sixty-two first-episode patients and 76 healthy control subjects, matched for age and premorbid IQ, were tested; 104 patients and 25 control subjects were reassessed 1 and 3 years later, and 31 patients were reassessed additionally 6 years later. RESULTS: Patients showed impaired set-shifting that correlated with current IQ and working memory, but there were no impairments when subgroups were matched on current IQ. In contrast, patients showed marked impairments in rule reversal learning that survived correction for IQ, were present in the context of intact rule abstraction, and correlated with disorganization symptoms. Patients prescribed second-generation antipsychotics were worse on set-shifting compared with first-generation, a finding not explained by demographic data, illness characteristics, or IQ. Patients and control subjects showed stable IDED performance over the first 6 years of illness, although set-shifting was inconsistent over the first year. Those with residual negative symptoms were more likely to fail the set-shifting stage at follow-up. CONCLUSIONS: First-episode schizophrenia patients can learn and generalize rules but are inflexible when rules change, reflecting reduced responsiveness to negative feedback and difficulty in switching attention. Rule-reversal is a promising target for translational studies, because it is specific, clinically relevant, and might reflect orbitofrontal dysfunction. Set-shifting is related to poor function more generally but might be sensitive to medication effects and valuable for clinical trials.
Asunto(s)
Atención/fisiología , Aprendizaje Discriminativo/fisiología , Discapacidades para el Aprendizaje/etiología , Esquizofrenia/complicaciones , Psicología del Esquizofrénico , Adolescente , Adulto , Antipsicóticos/uso terapéutico , Estudios de Casos y Controles , Retroalimentación Psicológica/fisiología , Femenino , Humanos , Inteligencia , Estudios Longitudinales , Masculino , Memoria a Corto Plazo/fisiología , Pruebas Neuropsicológicas , Solución de Problemas/fisiología , Escalas de Valoración Psiquiátrica , Esquizofrenia/tratamiento farmacológico , Estadísticas no Paramétricas , Factores de Tiempo , Adulto JovenRESUMEN
The search for target genes involved in unbalanced acquired chromosomal abnormalities has been largely unsuccessful, because the breakpoints of these rearrangements are too variable. Here, we use the example of dicentric chromosomes in B cell precursor acute lymphoblastic leukemia to show that, despite this heterogeneity, single genes are targeted through a variety of mechanisms. FISH showed that, although they were heterogeneous, breakpoints on 9p resulted in the partial or complete deletion of PAX5. Molecular copy number counting further delineated the breakpoints and facilitated cloning with long-distance inverse PCR. This approach identified 5 fusion gene partners with PAX5: LOC392027 (7p12.1), SLCO1B3 (12p12), ASXL1 (20q11.1), KIF3B (20q11.21), and C20orf112 (20q11.1). In each predicted fusion protein, the DNA-binding paired domain of PAX5 was present. Using quantitative PCR, we demonstrated that both the deletion and gene fusion events resulted in the same underexpression of PAX5, which extended to the differential expression of the PAX5 target genes, EBF1, ALDH1A1, ATP9A, and FLT3. Further molecular analysis showed deletion and mutation of the homologous PAX5 allele, providing further support for the key role of PAX5. Here, we show that specific gene loci may be the target of heterogeneous translocation breakpoints in human cancer, acting through a variety of mechanisms. This approach indicates an application for the identification of cancer genes in solid tumours, where unbalanced chromosomal rearrangements are particularly prevalent and few genes have been identified. It can be extrapolated that this strategy will reveal that the same mechanisms operate in cancer pathogenesis in general.
Asunto(s)
Rotura Cromosómica , Genes Relacionados con las Neoplasias , Factor de Transcripción PAX5/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Translocación Genética/genética , Alelos , Secuencia de Bases , Cromosomas Humanos Par 9/genética , Dosificación de Gen , Humanos , Hibridación Fluorescente in Situ , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Proteínas de Fusión Oncogénica/genética , Factor de Transcripción PAX5/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismoRESUMEN
Deregulation of the RAS-RAF-mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK signaling cascade is often caused by somatic mutations in genes encoding proteins which influence the activity of this pathway and include NRAS, KRAS2, FLT3, PTPN11, and BRAF. We report the first comprehensive mutational screen of key exons of these genes in a large cohort of unselected acute lymphoblastic leukemia (ALL) cases at diagnosis (n = 86) and in a more selected cohort at disease recurrence (n = 47) using the sensitive method of denaturing high-performance liquid chromatography. We show that somatic mutations that deregulate the pathway constitute one of the most common genetic aberrations in childhood ALL (cALL), being found in 35% of diagnostic and 25% of relapse samples. In matched presentation/relapse pairs, mutations predominating at relapse could be shown to be present at very low levels at diagnosis using allele-specific PCR, thus implicating the mutated clone in disease progression. Importantly, in primary samples, we show that mutations are associated with activated ERK and differential cytotoxicity to MEK-ERK inhibitors was shown for some patients. Inhibitors of the pathway, which are currently undergoing clinical trial, may be a novel therapeutic option for cALL, particularly at relapse.
Asunto(s)
Genes ras/fisiología , Mutación/genética , Recurrencia Local de Neoplasia/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogénicas/genética , Transducción de Señal , Proteínas ras/genética , Adolescente , Western Blotting , Supervivencia Celular , Niño , Preescolar , Estudios de Cohortes , Análisis Mutacional de ADN , Inhibidores Enzimáticos/farmacología , Exones/genética , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Hibridación Fluorescente in Situ , Lactante , Masculino , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Recurrencia Local de Neoplasia/patología , Fragmentos de Péptidos , Ploidias , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras) , Inducción de Remisión , Células Tumorales Cultivadas , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
The MSH3 and dihydrofolate reductase (DHFR) genes, located on chromosome 5, share a common promoter but are divergently transcribed. Dysregulation of the mismatch repair (MMR) pathway has been found to occur in cell line models due to co-amplification of MSH3 as a coincident effect of DHFR amplification, acquired as a mechanism generating resistance to methotrexate (MTX). The increased levels of MSH3 perturbed MutSalpha function resulting in hypermutability and increased resistance to thiopurines, drugs whose cytotoxic effects are triggered by MutSalpha. The relevance of this phenomenon in clinical samples is unknown but is extremely pertinent in childhood acute lymphoblastic leukaemia (ALL) in which children are exposed for prolonged periods to both MTX and thiopurines such that a single amplification event involving both the DHFR and the MSH3 genes may cause chemotherapeutic resistance to both agents. Thus, we have generated a leukaemic cell line (PreB697) and a normal human lymphoblastoid cell line (TK6) that are resistant to a pharmacologically relevant dose of MTX and show that while increased DHFR levels result in MTX resistance, the associated increased levels of MSH3 are insufficient to perturb MutSalpha functionality, in terms of MMR capacity or 6-thioguanine sensitivity. In addition, we show that although low-level DHFR amplification occurs alone in a significant number of samples, both at disease onset and relapse, co-amplification of both MSH3 and DHFR is rarely found in primary ALL samples, even after prolonged MTX therapy and is not at a sufficiently high level to perturb MMR function.
Asunto(s)
Proteínas de Unión al ADN/genética , Amplificación de Genes/fisiología , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimología , Tetrahidrofolato Deshidrogenasa/genética , Línea Celular Transformada , Línea Celular Tumoral , Niño , Proteínas de Unión al ADN/metabolismo , Dosificación de Gen , Humanos , Metotrexato/administración & dosificación , Metotrexato/farmacología , Proteína 3 Homóloga de MutS , Recurrencia Local de Neoplasia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Tetrahidrofolato Deshidrogenasa/metabolismoRESUMEN
Glucocorticoids (GCs) specifically induce apoptosis in malignant lymphoblasts and are thus pivotal in the treatment of acute lymphoblastic leukemia (ALL). However, GC-resistance is a therapeutic problem with an unclear molecular mechanism. We generated approximately 70 GC-resistant sublines from a GC-sensitive B- and a T-ALL cell line and investigated their mechanisms of resistance. In response to GCs, all GC-resistant subclones analyzed by real-time polymerase chain reaction (PCR) showed a deficient up-regulation of the GC-receptor (GR) and its downstream target, GC-induced leucine zipper. This deficiency in GR up-regulation was confirmed by Western blotting and on retroviral overexpression of GR in resistant subclones GC-sensitivity was restored. All GC-resistant subclones were screened for GR mutations using denaturing high-pressure liquid chromatography (DHPLC), DNA-fingerprinting, and fluorescence in situ hybridization (FISH). Among the identified mutations were some previously not associated with GC resistance: A484D, P515H, L756N, Y663H, L680P, and R714W. This approach revealed three genotypes, complete loss of functional GR in the mismatch repair deficient T-ALL model, apparently normal GR genes in B-ALLs, and heterozygosity in both. In the first genotype, deficiency in GR up-regulation was fully explained by mutational events, in the second by a putative regulatory defect, and in the third by a combination thereof. In all instances, GC-resistance occurred at the level of the GR in both models.
Asunto(s)
Resistencia a Antineoplásicos , Glucocorticoides/uso terapéutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Receptores de Glucocorticoides/metabolismo , Línea Celular Tumoral , Reparación de la Incompatibilidad de ADN , Resistencia a Antineoplásicos/genética , Glucocorticoides/metabolismo , Humanos , Mutación , Receptores de Glucocorticoides/genética , Factores de Transcripción/metabolismoRESUMEN
Defects in the mismatch repair system are associated with a microsatellite unstable phenotype. In this chapter, we describe the preparation of purified plasma cells using CD138 magnetic microbeads as a source of tumor DNA. We also describe a robust, sensitive method for comparing microsatellite repeat units of tumor to constitutive DNA using polymerase chain reaction and laser scanning of fluorescently labeled amplicons in an automated sequencer in order to assess microsatellite instability in myeloma.
Asunto(s)
Disparidad de Par Base/genética , Mieloma Múltiple/genética , Antígenos CD/genética , Secuencia de Bases , Cartilla de ADN , ADN de Neoplasias/genética , ADN de Neoplasias/aislamiento & purificación , Electroforesis Capilar/métodos , Humanos , Glicoproteínas de Membrana/genética , Repeticiones de Microsatélite , Mieloma Múltiple/inmunología , Reacción en Cadena de la Polimerasa/métodos , Proteoglicanos/genética , Sindecano-1 , SindecanosRESUMEN
Genetic instability is a prominent feature in multiple myeloma and progression of this disease from monoclonal gammopathy of uncertain significance (MGUS) and smouldering myeloma (SMM) is associated with increasing molecular and chromosomal abnormalities. The DNA mismatch repair (MMR) pathway is a post-replicational DNA repair system that maintains genetic stability by repairing mismatched bases and insertion/deletion loops mistakenly incorporated during DNA replication. Deficiencies in proteins pivotal to this pathway result in a higher mutation rate, particularly at regions of microsatellite DNA. We have investigated the proficiency of the MMR pathway in clinical samples and myeloma cell lines. Microsatellite analysis showed instability at one or more of nine loci examined in 15 from 92 patients: 7.7% of MGUS/SMM, 20.7% of MM/plasma cell leukaemia (PCL) and 12.5% of relapsed MM/PCL. An in vitro heteroduplex G/T repair assay found reduced repair in two cell lines, JIM1 and JIM3, and in two of four PCL cases and was associated with aberrant expression of at least one mismatch repair protein. Thus we show that MMR defects are found in plasma cell dyscrasias and the increased frequency during more active stages of the disease suggests a contributory role in disease progression.
Asunto(s)
Disparidad de Par Base , Reparación del ADN , Proteínas de Unión al ADN/genética , Mieloma Múltiple/genética , Proteínas de Neoplasias/genética , Proteínas Adaptadoras Transductoras de Señales , Adulto , Anciano , Anciano de 80 o más Años , Proteínas Portadoras , Daño del ADN , Metilación de ADN , Replicación del ADN , ADN de Neoplasias/genética , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Repeticiones de Microsatélite , Persona de Mediana Edad , Homólogo 1 de la Proteína MutL , Proteínas NuclearesRESUMEN
Mitochondrial DNA (mtDNA) defects cause debilitating metabolic disorders for which there is no effective treatment. Patients suffering from these diseases often harbour both a wild-type and a mutated subpopulation of mtDNA, a situation termed heteroplasmy. Understanding mtDNA repair mechanisms could facilitate the development of novel therapies to combat these diseases. In particular, mismatch repair activity could potentially be used to repair pathogenic mtDNA mutations existing in the heteroplasmic state if heteroduplexes could be generated. To date, however, there has been no compelling evidence for such a repair activity in mammalian mitochondria. We now report evidence consistent with a mismatch repair capability in mammalian mitochondria that exhibits some characteristics of the nuclear pathway. A repair assay utilising a nicked heteroduplex substrate with a GT or a GG mismatch in the beta-galactosidase reporter gene was used to test the repair potential of different lysates. A low level repair activity was identified in rat liver mitochondrial lysate that showed no strand bias. The activity was mismatch-selective, bi-directional, ATP-dependent and EDTA-sensitive. Western analysis using antibody to MSH2, a key nuclear mismatch repair system (MMR) protein, showed no cross-reacting species in mitochondrial lysate. A hypothesis to explain the molecular mechanism of mitochondrial MMR in the light of these observations is discussed.