RESUMEN
X-linked inhibitor of apoptosis (XIAP) deficiency, caused by mutations in BIRC4, is an immunodeficiency associated with immune dysregulation and a highly variable clinical presentation. Current diagnostic screening tests such as flow cytometry for XIAP expression or lymphocyte apoptosis assays have significant limitations. Based on recent evidence that XIAP is essential for nucleotide-binding and oligomerization domains (NOD)1/2 signalling, we evaluated the use of a simple flow cytometric assay assessing tumour necrosis factor (TNF) production of monocytes in response to NOD2 stimulation by muramyl dipeptides (L18-MDP) for the functional diagnosis of XIAP deficiency. We investigated 12 patients with XIAP deficiency, six female carriers and relevant disease controls. Irrespective of the diverse clinical phenotype, the extent of residual protein expression or the nature of the mutation, the TNF response was severely reduced in all patients. On average, L18-MDP induced TNF production in 25% of monocytes from healthy donors or female carriers, while fewer than 6% of monocytes responded in affected patients. Notably, the assay clearly discriminated affected patients from disease controls with other immunodeficiencies accompanied by lymphoproliferation, hypogammaglobulinaemia or inflammatory bowel disease. Functional testing of the NOD2 signalling pathway is an easy, fast and reliable assay in the diagnostic evaluation of patients with suspected XIAP deficiency.
Asunto(s)
Síndromes de Inmunodeficiencia/diagnóstico , Síndromes de Inmunodeficiencia/genética , Proteína Inhibidora de la Apoptosis Ligada a X/deficiencia , Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Acetilmuramil-Alanil-Isoglutamina/farmacología , Adolescente , Adulto , Anciano , Estudios de Casos y Controles , Niño , Preescolar , Femenino , Citometría de Flujo , Humanos , Inmunofenotipificación , Lactante , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Mutación , Proteína Adaptadora de Señalización NOD2/metabolismo , Fenotipo , Linfocitos T/metabolismo , Factores de Necrosis Tumoral/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Adulto JovenRESUMEN
X-linked inhibitor of apoptosis (XIAP) deficiency caused by mutations in BIRC4 was initially described in patients with X-linked lymphoproliferative syndrome (XLP) who had no mutations in SH2D1A. In the initial reports, EBV-associated hemophagocytic lymphohistiocytosis (HLH) was the predominant clinical phenotype. Among 25 symptomatic patients diagnosed with XIAP deficiency, we identified 17 patients who initially presented with manifestations other than HLH. These included Crohn-like bowel disease (n=6), severe infectious mononucleosis (n=4), isolated splenomegaly (n=3), uveitis (n=1), periodic fever (n=1), fistulating skin abscesses (n=1) and severe Giardia enteritis (n=1). Subsequent manifestations included celiac-like disease, antibody deficiency, splenomegaly and partial HLH. Screening by flow cytometry identified 14 of 17 patients in our cohort. However, neither genotype nor protein expression nor results from cell death studies were clearly associated with the clinical phenotype. Only mutation analysis can reliably identify affected patients. XIAP deficiency must be considered in a wide range of clinical presentations.
Asunto(s)
Síndromes de Inmunodeficiencia/genética , Linfohistiocitosis Hemofagocítica/genética , Proteína Inhibidora de la Apoptosis Ligada a X/deficiencia , Adolescente , Adulto , Niño , Preescolar , Genotipo , Humanos , Síndromes de Inmunodeficiencia/inmunología , Linfohistiocitosis Hemofagocítica/inmunología , Masculino , Mutación , Células T Asesinas Naturales/inmunología , Fenotipo , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/inmunología , Adulto JovenRESUMEN
Chromosomal rearrangements of the human KMT2A/MLL gene are associated with de novo as well as therapy-induced infant, pediatric, and adult acute leukemias. Here, we present the data obtained from 3401 acute leukemia patients that have been analyzed between 2003 and 2022. Genomic breakpoints within the KMT2A gene and the involved translocation partner genes (TPGs) and KMT2A-partial tandem duplications (PTDs) were determined. Including the published data from the literature, a total of 107 in-frame KMT2A gene fusions have been identified so far. Further 16 rearrangements were out-of-frame fusions, 18 patients had no partner gene fused to 5'-KMT2A, two patients had a 5'-KMT2A deletion, and one ETV6::RUNX1 patient had an KMT2A insertion at the breakpoint. The seven most frequent TPGs and PTDs account for more than 90% of all recombinations of the KMT2A, 37 occur recurrently and 63 were identified so far only once. This study provides a comprehensive analysis of the KMT2A recombinome in acute leukemia patients. Besides the scientific gain of information, genomic breakpoint sequences of these patients were used to monitor minimal residual disease (MRD). Thus, this work may be directly translated from the bench to the bedside of patients and meet the clinical needs to improve patient survival.
Asunto(s)
N-Metiltransferasa de Histona-Lisina , Leucemia Mieloide Aguda , Proteína de la Leucemia Mieloide-Linfoide , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Lactante , Preescolar , Niño , Adolescente , Adulto , Persona de Mediana Edad , Anciano , Leucemia Mieloide Aguda/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , N-Metiltransferasa de Histona-Lisina/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Fusión GénicaRESUMEN
Two related boys who died from fulminant infectious mononucleosis were diagnosed with X-linked lymphoproliferative disease type 1 (XLP-1). Family screening (n=17) identified 6 female mutation carriers and 2 more XLP-1 patients in whom, despite recurrent infections, agammaglobulinemia, and Hodgkin's Disease, the genetic basis had been unknown; demonstrating that awareness and early genetic testing are crucial to reveal underlying primary immunodeficiencies and improve outcome. Furthermore, XLP should be included routinely in the differential diagnosis of severe hypogammaglobulinemia and/or lymphoma in males.
Asunto(s)
Mononucleosis Infecciosa/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Linfohistiocitosis Hemofagocítica/genética , Trastornos Linfoproliferativos/genética , Adolescente , Agammaglobulinemia/diagnóstico , Agammaglobulinemia/genética , Preescolar , Análisis Mutacional de ADN , Infecciones por Virus de Epstein-Barr/diagnóstico , Infecciones por Virus de Epstein-Barr/genética , Exones/genética , Resultado Fatal , Tamización de Portadores Genéticos , Enfermedades Genéticas Ligadas al Cromosoma X/diagnóstico , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Pruebas Genéticas , Enfermedad de Hodgkin/diagnóstico , Enfermedad de Hodgkin/genética , Humanos , Lactante , Mononucleosis Infecciosa/diagnóstico , Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/genética , Linfohistiocitosis Hemofagocítica/diagnóstico , Linfohistiocitosis Hemofagocítica/mortalidad , Trastornos Linfoproliferativos/diagnóstico , Trastornos Linfoproliferativos/mortalidad , Masculino , Meningoencefalitis/complicaciones , Meningoencefalitis/diagnóstico , Meningoencefalitis/genética , Mutación Missense , Linaje , Proteína Asociada a la Molécula de Señalización de la Activación Linfocitaria , Adulto JovenRESUMEN
BACKGROUND: PRF1 gene mutations are associated with familial haemophagocytic lymphohistiocytosis type 2 (FHL2). Genotype-phenotype analysis, previously hampered by limited numbers of patients, was for the first time performed by data pooling from five large centres worldwide. PATIENTS AND METHODS: Members of the Histiocyte Society were asked to report cases of FHL2 on specific forms. Data were pooled in a common database and analysed. RESULTS: The 124 patients had 63 different mutations (including 15 novel mutations): 11 nonsense, 10 frameshift, 38 missense and 4 in-frame deletions. Some mutations were found more commonly: 1122 G-->A (W374X), associated with Turkish origin, in 32 patients; 50delT (L17fsX22) associated with African/African American origin, in 21 patients; and 1090-91delCT (L364fsX), in 7 Japanese patients. Flow cytometry showed that perforin expression was absent in 40, reduced in 6 and normal in 4 patients. Patients presented at a median age of 3 months (quartiles: 2, 3 and 13 months), always with fever, splenomegaly and thrombocytopenia. NK activity was absent in 36 (51%),
Asunto(s)
Linfohistiocitosis Hemofagocítica/etnología , Linfohistiocitosis Hemofagocítica/fisiopatología , Mutación , Perforina/genética , Adolescente , Adulto , Niño , Preescolar , Etnicidad , Femenino , Mutación del Sistema de Lectura , Genotipo , Humanos , Lactante , Recién Nacido , Células Asesinas Naturales/inmunología , Linfohistiocitosis Hemofagocítica/genética , Linfohistiocitosis Hemofagocítica/inmunología , Masculino , Mutación Missense , FenotipoRESUMEN
Most modern treatment protocols for acute lymphoblastic leukaemia (ALL) include the analysis of minimal residual disease (MRD). To ensure comparable MRD results between different MRD-polymerase chain reaction (PCR) laboratories, standardization and quality control are essential. The European Study Group on MRD detection in ALL (ESG-MRD-ALL), consisting of 30 MRD-PCR laboratories worldwide, has developed guidelines for the interpretation of real-time quantitative PCR-based MRD data. The application of these guidelines ensures identical interpretation of MRD data between different laboratories of the same MRD-based clinical protocol. Furthermore, the ESG-MRD-ALL guidelines will facilitate the comparison of MRD data obtained in different treatment protocols, including those with new drugs.
Asunto(s)
Reordenamiento Génico , Neoplasia Residual/genética , Reacción en Cadena de la Polimerasa/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptores de Antígenos de Linfocitos T/genética , ADN de Neoplasias/genética , Genes de Inmunoglobulinas , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/microbiologíaRESUMEN
Chromosomal rearrangements of the human MLL/KMT2A gene are associated with infant, pediatric, adult and therapy-induced acute leukemias. Here we present the data obtained from 2345 acute leukemia patients. Genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and 11 novel TPGs were identified. Thus, a total of 135 different MLL rearrangements have been identified so far, of which 94 TPGs are now characterized at the molecular level. In all, 35 out of these 94 TPGs occur recurrently, but only 9 specific gene fusions account for more than 90% of all illegitimate recombinations of the MLL gene. We observed an age-dependent breakpoint shift with breakpoints localizing within MLL intron 11 associated with acute lymphoblastic leukemia and younger patients, while breakpoints in MLL intron 9 predominate in AML or older patients. The molecular characterization of MLL breakpoints suggests different etiologies in the different age groups and allows the correlation of functional domains of the MLL gene with clinical outcome. This study provides a comprehensive analysis of the MLL recombinome in acute leukemia and demonstrates that the establishment of patient-specific chromosomal fusion sites allows the design of specific PCR primers for minimal residual disease analyses for all patients.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/genética , Leucemia Mieloide Aguda/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Adulto , Niño , Aberraciones Cromosómicas , Rotura Cromosómica , Femenino , Reordenamiento Génico/genética , Humanos , Lactante , Masculino , Proteínas de Fusión Oncogénica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Translocación Genética/genéticaRESUMEN
Chromosomal rearrangements of the human MLL gene are a hallmark for aggressive (high-risk) pediatric, adult and therapy-associated acute leukemias. These patients need to be identified in order to subject these patients to appropriate therapy regimen. A recently developed long-distance inverse PCR method was applied to genomic DNA isolated from individual acute leukemia patients in order to identify chromosomal rearrangements of the human MLL gene. We present data of the molecular characterization of 414 samples obtained from 272 pediatric and 142 adult leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) was determined and several new TPGs were identified. The combined data of our study and published data revealed a total of 87 different MLL rearrangements of which 51 TPGs are now characterized at the molecular level. Interestingly, the four most frequently found TPGs (AF4, AF9, ENL and AF10) encode nuclear proteins that are part of a protein network involved in histone H3K79 methylation. Thus, translocations of the MLL gene, by itself coding for a histone H3K4 methyltransferase, are presumably not randomly chosen, rather functionally selected.
Asunto(s)
Leucemia/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Fusión Oncogénica/genética , Translocación Genética/genética , Enfermedad Aguda , Adulto , Niño , Aberraciones Cromosómicas , Mapeo Cromosómico , ADN/genética , ADN/aislamiento & purificación , N-Metiltransferasa de Histona-Lisina , Histonas/metabolismo , Humanos , MetilaciónRESUMEN
Children with P2RY8-CRLF2-positive acute lymphoblastic leukemia have an increased relapse risk. Their mutational and transcriptional landscape, as well as the respective patterns at relapse remain largely elusive. We, therefore, performed an integrated analysis of whole-exome and RNA sequencing in 41 major clone fusion-positive cases including 19 matched diagnosis/relapse pairs. We detected a variety of frequently subclonal and highly instable JAK/STAT but also RTK/Ras pathway-activating mutations in 76% of cases at diagnosis and virtually all relapses. Unlike P2RY8-CRLF2 that was lost in 32% of relapses, all other genomic alterations affecting lymphoid development (58%) and cell cycle (39%) remained stable. Only IKZF1 alterations predominated in relapsing cases (P=0.001) and increased from initially 36 to 58% in matched cases. IKZF1's critical role is further corroborated by its specific transcriptional signature comprising stem cell features with signs of impaired lymphoid differentiation, enhanced focal adhesion, activated hypoxia pathway, deregulated cell cycle and increased drug resistance. Our findings support the notion that P2RY8-CRLF2 is dispensable for relapse development and instead highlight the prominent rank of IKZF1 for relapse development by mediating self-renewal and homing to the bone marrow niche. Consequently, reverting aberrant IKAROS signaling or its disparate programs emerges as an attractive potential treatment option in these leukemias.
Asunto(s)
Fusión Génica , Genómica , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptores de Citocinas/genética , Receptores Purinérgicos P2Y/genética , Transcripción Genética , Adolescente , Niño , Preescolar , Dosificación de Gen , Genes Supresores de Tumor , Humanos , Factor de Transcripción Ikaros/genética , Factor de Transcripción Ikaros/fisiología , Lactante , Quinasas Janus/fisiología , Polimorfismo de Nucleótido Simple , Factores de Transcripción STAT/fisiologíaRESUMEN
High hyperdiploidy defines the largest genetic entity of childhood acute lymphoblastic leukemia (ALL). Despite its relatively low recurrence risk, this subgroup generates a high proportion of relapses. The cause and origin of these relapses remains obscure. We therefore explored the mutational landscape in high hyperdiploid (HD) ALL with whole-exome (n=19) and subsequent targeted deep sequencing of 60 genes in 100 relapsing and 51 non-relapsing cases. We identified multiple clones at diagnosis that were primarily defined by a variety of mutations in receptor tyrosine kinase (RTK)/Ras pathway and chromatin-modifying genes. The relapse clones consisted of reappearing as well as new mutations, and overall contained more mutations. Although RTK/Ras pathway mutations were similarly frequent between diagnosis and relapse, both intergenic and intragenic heterogeneity was essentially lost at relapse. CREBBP mutations, however, increased from initially 18-30% at relapse, then commonly co-occurred with KRAS mutations (P<0.001) and these relapses appeared primarily early (P=0.012). Our results confirm the exceptional susceptibility of HD ALL to RTK/Ras pathway and CREBBP mutations, but, more importantly, suggest that mutant KRAS and CREBBP might cooperate and equip cells with the necessary capacity to evolve into a relapse-generating clone.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Proteína de Unión a CREB/genética , Diploidia , Mutación/genética , Recurrencia Local de Neoplasia/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogénicas/genética , Proteínas ras/genética , Adolescente , Estudios de Casos y Controles , Niño , Evolución Clonal , Femenino , Estudios de Seguimiento , Humanos , Masculino , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/mortalidad , Recurrencia Local de Neoplasia/patología , Estadificación de Neoplasias , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidad , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Pronóstico , Proteínas Proto-Oncogénicas p21(ras) , Tasa de SupervivenciaAsunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasia Residual , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Resultado del Tratamiento , Niño , Humanos , Inmunofenotipificación , Reacción en Cadena de la Polimerasa , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , PronósticoAsunto(s)
Histiocitosis de Células no Langerhans/genética , Glicoproteínas de Membrana/genética , Mutación/genética , Niño , Preescolar , Codón sin Sentido/genética , Codón de Terminación/genética , Consanguinidad , Análisis Mutacional de ADN , Exones/genética , Femenino , Ghana , Histiocitosis de Células no Langerhans/patología , Humanos , Lactante , Italia , Masculino , Glicoproteínas de Membrana/química , Mutación Missense/genética , Perforina , Proteínas Citotóxicas Formadoras de Poros , Estructura Terciaria de Proteína , Resultado del Tratamiento , TurquíaRESUMEN
Primary hemophagocytic syndromes represent a group of rare immunodeficiencies, which are characterized by development of life-threatening systemic inflammatory manifestations, so-called accelerated phases. Immunosuppressive therapies are only temporarily effective to control this complication and the prognosis is dismal unless treated by hematopoietic SCT (HSCT). At present, optimal modalities of this potentially curative approach remain incompletely defined. In this study, we analyzed our experience in 18 patients with primary hemophagocytic syndromes treated since 1984 in our center by HSCT. Ten of these patients had previously developed accelerated phases and were in remission at the time of HSCT, whereas five patients had findings of active disease, with two cases in early phases of recurrences of less than 2 weeks duration and three cases with persistent central nervous system disease, whereas three patients had never experienced accelerated phases. In the group with active disease, four of five patients are long-term survivors and are well, whereas one patient died of CMV pneumonia. This outcome compares favorably with results in patients transplanted in remission, where 6 of 10 are long-term survivors. Our findings indicate that HSCT can have a favorable prognosis even in patients with active disease of primary hemophagocytic syndrome.
Asunto(s)
Enfermedades del Sistema Nervioso Central/terapia , Síndrome de Chediak-Higashi/terapia , Trasplante de Células Madre Hematopoyéticas , Hipopigmentación/terapia , Síndromes de Inmunodeficiencia/terapia , Linfohistiocitosis Hemofagocítica/terapia , Acondicionamiento Pretrasplante , Enfermedades del Sistema Nervioso Central/prevención & control , Niño , Preescolar , Quimerismo , Progresión de la Enfermedad , Femenino , Enfermedad Injerto contra Huésped/complicaciones , Reacción Injerto-Huésped , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Trasplante de Células Madre Hematopoyéticas/mortalidad , Humanos , Hipopigmentación/complicaciones , Hipopigmentación/prevención & control , Síndromes de Inmunodeficiencia/complicaciones , Síndromes de Inmunodeficiencia/prevención & control , Lactante , Recién Nacido , Depleción Linfocítica , Linfohistiocitosis Hemofagocítica/prevención & control , Masculino , Embarazo , Pronóstico , Recurrencia , Inducción de Remisión , Sobrevivientes , Factores de Tiempo , Resultado del TratamientoRESUMEN
Chromosomal rearrangements of the human MLL gene are associated with high-risk pediatric, adult and therapy-associated acute leukemias. These patients need to be identified, treated appropriately and minimal residual disease was monitored by quantitative PCR techniques. Genomic DNA was isolated from individual acute leukemia patients to identify and characterize chromosomal rearrangements involving the human MLL gene. A total of 760 MLL-rearranged biopsy samples obtained from 384 pediatric and 376 adult leukemia patients were characterized at the molecular level. The distribution of MLL breakpoints for clinical subtypes (acute lymphoblastic leukemia, acute myeloid leukemia, pediatric and adult) and fused translocation partner genes (TPGs) will be presented, including novel MLL fusion genes. Combined data of our study and recently published data revealed 104 different MLL rearrangements of which 64 TPGs are now characterized on the molecular level. Nine TPGs seem to be predominantly involved in genetic recombinations of MLL: AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, MLLT4/AF6, ELL, EPS15/AF1P, MLLT6/AF17 and SEPT6, respectively. Moreover, we describe for the first time the genetic network of reciprocal MLL gene fusions deriving from complex rearrangements.
Asunto(s)
Leucemia/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Neoplasias/genética , Proteínas de Fusión Oncogénica/genética , Recombinación Genética , Translocación Genética , Enfermedad Aguda , Adulto , Biopsia , Médula Ósea/química , Médula Ósea/patología , Niño , Rotura Cromosómica , Cromosomas Humanos Par 11/genética , Cromosomas Humanos Par 11/ultraestructura , Biología Computacional , ADN de Neoplasias/sangre , ADN de Neoplasias/genética , Duplicación de Gen , N-Metiltransferasa de Histona-Lisina , Humanos , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: Denaturing HPLC (DHPLC) can be used to screen DNA for known and unknown mutations. We describe a novel, HPLC-based method for discrimination among polyclonal, oligoclonal, and/or clonal T-cell receptor gamma (TCR-gamma) rearrangements in samples from children with newly diagnosed acute lymphoblastic leukemia. METHODS: TCR rearrangements were PCR amplified from initial leukemic samples and, after heteroduplex-induction, the clonality status of each product was evaluated. To attain this, we used DHPLC on a high-resolution micropellicular matrix. Running conditions were established by melting-curve analysis of known clonal and polyclonal products and melting-point prediction software. Elution profiles were studied at 50 degrees C (native) and, to achieve optimal separation, at different column temperatures between 56 and 64 degrees C. RESULTS: For VgammaI-Jgamma1.3/2.3 rearrangements, an analysis temperature of 60 degrees C with a linear triethylammoniumacetate-acetonitrile gradient separated clonal bands from the polyclonal background amplification. Less than 15% clonal PCR product was detectable in mixtures of initial leukemic cell DNA and polyclonal DNA. Biallelic rearrangements produced two sharp peaks. Clonality of PCR products from 100 initial leukemic samples was completely identified in all investigated cases. CONCLUSIONS: Heteroduplex analysis with standardized DHPLC conditions simplifies the detection of unknown clonal or polyclonal TCR rearrangements in newly diagnosed leukemias. Clonal targets for detection of minimal residual disease are available after a short, automated analysis of PCR amplified rearrangements.
Asunto(s)
Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Secuencia de Bases , Cromatografía Líquida de Alta Presión/métodos , Análisis Heterodúplex , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
Interleukin-6 (IL-6) is a cytokine which is necessary for the differentiation of activated B cells and growth of early haemopoietic progenitors. It is used for ex-vivo expansion of myeloid progenitors in the setting of high-dose chemotherapy with autologous bone marrow transplantation (BMT). Expression of the IL-6 receptor (IL-6R) was examined in six fresh Burkitt's lymphoma (BL) cell preparations and 12 BL cell lines by reverse transcriptase polymerase chain reaction (RT-PCR) and flow cytometry (FCM). Inducibility of IL-6R mRNA expression by Epstein-Barr virus (EBV) was studied by comparing two uninfected cell lines with the same cell lines infected by EBV The phenotype of the BL cells lines was analysed by FCM and by proliferation assays in the presence of anti-IgM antibodies. None of the fresh BL cells expressed the IL-6 receptor. The BL cell lines expressed varying degrees of IL-6R mRNA and protein. In vitro infection of EBV-negative BL cell lines resulted in up-regulation of IL-6R mRNA. There was no proliferative response of the BL cell lines to IL-6, including the cells that expressed the receptor. Compared to uninfected BL cell lines, EBV-infected cell lines and lymphoblastoid cell lines (LCLs) showed a weaker or no response to anti-IgM antibodies, indicating a more mature phenotype of these cells. We conclude that the IL-6 receptor is not expressed in fresh childhood BLs, but only in BL cell lines. EBV infection in vitro leads to an up-regulation of IL-6R mRNA but not to increased proliferation. This makes growth stimulation of contaminating BL cells in the setting of autologous BMT unlikely.
Asunto(s)
Antígenos CD/metabolismo , Linfoma de Burkitt/metabolismo , Herpesvirus Humano 4/metabolismo , Receptores de Interleucina/metabolismo , División Celular , Niño , Citometría de Flujo , Humanos , Fenotipo , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Receptores de Interleucina-6 , Células Tumorales CultivadasRESUMEN
BACKGROUND: Burkitt's lymphoma (BL) and B-ALL are characterized by chromosomal translocations juxtaposing the c-myc gene on chromosome 8 to one of the immunoglobulin loci. Translocations involving the immunoglobulin heavy chain (IgH) on chromosome 14 are found in approximately 75%-90% of these tumors. The breakpoint regions are located over a wide range on both chromosomes. PATIENTS AND METHODS: To detect the translocations, we developed a PCR method to generate long products. After extraction of genomic DNA (QiaAmp System, Qiagen, Hilden, Germany), DNA was amplified using a mixture of Taq and Pwo polymerases (Boehringer Mannheim, Germany). Several primer pairs from the S mu, JH, CH1 and the C alpha regions on IgH and from exon 1 and intron 1 of the c-myc gene were tested in each patient. RESULTS: Lymphoma cells from 20 children with Burkitt's lymphoma and B-ALL characterized by FAB-L3 morphology were examined. In 11/20 patients, recombinations between chromosomes 8 and 14 could be detected with our primer pairs. PCR products from 800 to 3700 bp in length were obtained reproducibly. After amplification, the products were characterized by restriction enzyme digestion, hybridization, and in part by direct sequencing. CONCLUSIONS: This PCR-based method will allow us (1) to determine the localization of chromosomal breakpoints in primary tumor material, (2) to investigate whether distinct breakpoints are associated with treatment outcome, and (3) to detect the presence of minimal residual tumor cells during or after therapy.
Asunto(s)
Linfoma de Burkitt/genética , Cromosomas Humanos Par 14 , Cromosomas Humanos Par 8 , Reacción en Cadena de la Polimerasa/métodos , Translocación Genética , Adolescente , Niño , Preescolar , Rotura Cromosómica , Cartilla de ADN , ADN de Neoplasias/análisis , ADN de Neoplasias/genética , Exones , Femenino , Genes myc , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Intrones , Masculino , Oligonucleótidos/análisis , Oligonucleótidos/genética , Mapeo RestrictivoRESUMEN
The pediatric Non-Hodgkin's lymphomas are a heterogeneous group of malignancies of B- or T-cell origin. Approximately half of them are characterized as Burkitt's lymphomas. Typically, one of the reciprocal translocations t(8;14)(q24;q32), t(2;8)(p11;q24) or t(8;22)(q24;q11) is seen in the tumor cell, each involving the protooncogene c-myc on chromosome 8. Characteristically, in most patients the translocation occurs between the distal end of the long arm on chromosome 8 (c-myc) and chromosome 14 (immunoglobulin heavy chain locus, IgH). The breakpoint regions are distributed over a wide range of more than 10 Kb on chr. 8 and over several hundred Kb on chr. 14. With standard-PCR, fragments can only be amplified to a size of about 2 Kb. The development of PCR-applications to generate long products up to 20 Kb now allows a detection of these breakpoints. Several primer pairs from different regions of the IgH-gene and the c-myc-gene were tested in each patient. Until now, 20 patients with Burkitt's Lymphoma or B-ALL characterized by L3 morphology were examined. All patients were treated according the protocols of the NHL-BFM '90 or '95 study. In 11/20 patients, recombinations between chromosomes 8 and 14 could be detected with our primer pairs. In serial dilutions of DNA from malignant cells in DNA from healthy controls, sensitivities of one malignant cell in 2 x 10(4) normal cells could be obtained. This method will now allow us to characterize the involved breakpoints more exactly and to analyze patient samples (blood, bone marrow, aphereses products and residual tumors) during or after therapy for the existence of minimal residual tumor cells.
Asunto(s)
Linfoma de Burkitt/diagnóstico , Cromosomas Humanos Par 14 , Cromosomas Humanos Par 8 , Reacción en Cadena de la Polimerasa/métodos , Neoplasias de los Tejidos Blandos/diagnóstico , Translocación Genética/genética , Adolescente , Linfoma de Burkitt/genética , Niño , Preescolar , Mapeo Cromosómico , Cromosomas Humanos Par 11 , Cromosomas Humanos Par 2 , ADN de Neoplasias/genética , Femenino , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Masculino , Proteínas Proto-Oncogénicas c-myc/genética , Neoplasias de los Tejidos Blandos/genéticaRESUMEN
UNLABELLED: The detection of minimal residual disease (MRD) is a major prognostic factor for treatment in acute lymphoblastic leukemia (ALL) of childhood. Several groups showed the predictive value of MRD after 5 weeks of chemotherapy (at the end of induction therapy). Patients with more than 1 leukemic cells in 100 cells (> or = 10(-2)) at this time-point have a significantly higher relapse rate. The MRD measurement has been shown to be an independent prognostic factor at several time points in the BFM study (ALL-BFM 90) as well as in the EORTC study. The aim of our investigations was the detection of MRD at the end of induction therapy within the COALL studies which is different from the above studies. In the COALL studies, therapy starts with a 1 week DNR prephase (24 h infusion on day one) and i.th. MTX. Induction therapy consisted of 3 drugs over a period of 4 weeks (Prednisolone, Vincristine and Daunorubicin), asparaginase is given later in consolidation. At the end of induction therapy, bone marrow was obtained for cytomorphologic and molecular analysis. PATIENTS AND METHODS: We investigated bone marrow samples from 76 patients. All patients were in morphologic remission at the end. of induction therapy. For MRD analysis, DNA was isolated from bone marrow mononuclear cells. Clonal T-cell-receptor (TCR) or immunoglobulin gene (IgH) rearrangements were identified by PCR. Monoclonal products were either sequenced directly (TCR) or after excision from high resolution agarose gels. Subsequently patient-specific oligonucleotides for allele-specific PCR were generated. PCR analysis was performed with 1 microgram DNA for each reaction within a semiquantitative matter. This method reached sensitivities down to 10(-5). RESULTS: Eighty-four percent of the analysed samples were MRD positive at the end of induction therapy. 20 out of 76 patient samples (26%) were highly positive (> or = 10(-2)), 28 patients had levels of about 10(-3) (37%), 16 had levels around 10(-4) (21%) and 12 patients had no detectable residual cells (16%). All analysed 15 T-ALL patients had detectable residual disease at this timepoint. Until now, 5/20 patients with very high MRD level at the end of induction therapy suffered a relapse. DISCUSSION: Patients with very high MRD level at the end of induction therapy showed an elevated risk of relapse, but the predictive value is much poorer than for example in the BFM 90 MRD-study. We suggest, that a high MRD level at this timepoint results from a different induction therapy compared to the BFM 90 study. In the COALL studies asparaginase is given only after induction therapy to decrease the risk of thrombosis. We would like to conclude that this differences were compensated later during therapy as the event free survival of both studies is similar. In conclusion, an optimal information from MRD studies is strongly associated with the given therapy. Therefore we initiated an additional MRD time-point after the first chemotherapy block in consolidation.