RESUMEN
Diffuse large B-cell lymphoma (DLBCL) represents the most frequent type of aggressive lymphoma. Deletions of the CDKN2A locus, encoding the proteins CDKN2A (P16), P14ARF, and of the CDKN2B locus, encoding the protein CDKN2B (P15), affect one-third of DLBCL patients. Although other mechanisms that decrease gene expression have been reported, such as promoter methylation, the prognostic value of these mechanisms is still unclear. We studied the deletion and methylation status of these genes in 171 patients and correlated the genomic results with their mRNA expression level and clinical outcome. CDKN2A, P14ARF, and CDKN2B deletions were significantly correlated with decreased mRNA expression (P<0.0001, P<0.0001, and P=0.0148, respectively). P14ARF was methylated in only two patients (1.3%), whereas CDKN2A and CDKN2B were methylated in 36.7 and 31.4% of patients, respectively. Methylation levels greater than 25% were associated with decreased expression of CDKN2A (P=0.0169). CDKN2A and CDKN2B inactivation by deletion or methylation was observed in 42.7 and 37.4% of cases, respectively. Including P14ARF deletions, we identified an inactivating mechanism for at least one of these genes in 47% of patients. Although gene inactivation was not correlated with the international prognostic index, P14ARF and CDKN2B inactivation was significantly associated with shorter survival (P=0.0048 and P=0.0413, respectively), whereas CDKN2A was not (P=0.085). Low mRNA expression levels of these genes were correlated with the ABC phenotype. Furthermore, our results show that an inactivating methylation was more frequent in the GCB phenotype.
Asunto(s)
Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Metilación de ADN , Linfoma de Células B Grandes Difuso/clasificación , Linfoma de Células B Grandes Difuso/genética , Proteína p14ARF Supresora de Tumor/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Islas de CpG/genética , Femenino , Dosificación de Gen , Silenciador del Gen , Humanos , Linfoma de Células B Grandes Difuso/mortalidad , Masculino , Persona de Mediana Edad , Pronóstico , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Eliminación de Secuencia , Tasa de Supervivencia , Adulto JovenRESUMEN
Diffuse large B cell lymphoma represent the most frequent subtype of non Hodgkin's lymphoma, accounting for 30-40% of cases. Several studies have shown that CDKN2A and CDKN2B deletions are frequent in these lymphomas. These genes encode the P14ARF, CDKN2A, and CDKN2B proteins which play a key role in the control of the G1/S transition of the cell cycle. Using array CGH and a quantitative multiplex PCR method, we have previously identified such deletions in 36% of cases at diagnosis. Using a walking strategy to approach the breakpoints of these deletions we could identify the breakpoints junction of thirteen deletions in 11 patients and of two unbalanced translocation leading to a loss of these genes. A minimal common deleted region of 22.4 kb containing exon 1ß of CDKN2A encoding P14ARF and exon 2 of CDKN2B encoding CDKN2B was present in all cases but one. Analysis by quantitative RT PCR showed that the expression level of these genes was decreased in these patients as compared with non deleted cases and that this level was correlated with the deletion status of P14ARF, CDKN2A, and CDKN2B. Analysis of the breakpoint sequences showed that some of them were clustered within a few hundred base-pairs suggesting, even if we failed to identify any clear recombination prone sequences, that in these deletions the rearrangement results from non-random mechanisms.