Estudio de perfiles moleculares en pacientes con síndrome mielodisplásico

Los SÍNDROMES MIELODISPLÁSICOS (SMD) son una enfermedad heterogénea clonal de células madre hematopoyéticas. Se caracteriza por una medula ósea ineficaz que produce citopenias en sangre periférica. La severidad de estas citopenias y el riesgo incrementado a progresar a una leucemia mieloide aguda determinarán la evolución de la enfermedad y del paciente. Actualmente estos pacientes se clasifican de acuerdo con 7 subcategorías que establece la revisión del 2008 de la clasificación de la WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. La alta heterogeneidad de cada subcategoría ha hecho necesarios sistemas de puntuación, el más utilizado es el IPSS-R (International Prognostic Scoring System) e incluye 3 variables, las citopenias en sangre periférica, el porcentaje de blastos en médula ósea y el cariotipo. Nos permite evaluar la supervivencia global (SG) y la probabilidad de progresión a leucemia mieloide aguda. La hipótesis global del trabajo es que alteraciones detectadas mediante técnicas moleculares permiten mejorar la definición del pronóstico de pacientes con SMD. Para dilucidar la hipótesis general, se han estudiado tres grupos de pacientes con cariotipos distintos: pacientes con SMD y cariotipo monosómico y/o complejo, pacientes con el cromosoma 7 alterado mediante FISH y pacientes con deleción 5q [del(5q)]. Pacientes con cariotipo monosómico y con alteraciones en el cromosoma 7 se incluyen en el grupo de SMD de alto riesgo. En cambio, pacientes con del(5q), cuando esta se detecta de manera aislada, se incluyen en el grupo de SMD de bajo riesgo. La del(5q) en un cariotipo complejo pasa a ser de pronóstico desfavorable. Primero analizamos el efecto del cariotipo monosómico (MK). Estudiamos 1.054 pacientes con SMD y cariotipo alterado con el objetivo de describir la incidencia, características y pronóstico del MK, y su relación con la SG y el riesgo de progresión a LMA. Este estudio ha permitido determinar que el mal pronóstico asociado a los pacientes con MK, en el contexto de un cariotipo complejo, se debe a la complejidad en el cariotipo lo que les confiere un peor pronóstico, es decir peor SG y mayor riesgo de progresión a LMA. Segundo, estudiamos 820 pacientes por FISH con SMD, su cariotipo no mostró la monosomía 7 o la deleción 7q. El objetivo del estudio era determinar si la detección mediante FISH de estas alteraciones afectaba a su clasificación. El 5,2% de los casos fueron positivos, esto es de importante relevancia ya que la -7 o la del(7q) se relacionan con un impacto pronóstico negativo. Este estudio nos permitió detectar diferencias significativas en la SG de pacientes con riesgo morfológico intermedio (WHO 2008) FISH positivos y negativos. Debido a la relación de estas alteraciones del cromosoma 7 y un peor pronóstico, sería altamente recomendable analizar mediante FISH las alteraciones que no cumplan criterios de clonalidad.a El último grupo de pacientes que estudiamos presentaban la del(5q). Estudiamos un total de 228 pacientes con neoplasias mieloides (SMD y LMA) y del(5q). Mediante el estudio por secuenciación masiva del exoma pudimos determinar los 6 genes más mutados en nuestra serie: TP53, DNMT3A, CSNK1A1, SF3B1, PRPF8 y ASXL1. TP53 y DNMT3A presentaron un efecto negativo en la SG de los pacientes, pero sólo DNMT3A, retuvo la influencia independiente en la SG, en el análisis multivariado. El perfil mutacional de pacientes con del(5q) no se ha podido determinar. Estudios iniciales en pacientes con CSNK1A1 mutado, por su relación con una buena respuesta a la lenalidomida. Los pacientes con TP53 deberían ser controlados de manera frecuente, por el impacto negativo en la SG de mutaciones en este gen. Y, por último, mutaciones en DNMT3A deberían estudiarse en más pacientes con del(5q) para esclarecer su relación.MYELODYSPLASTIC SYNDROMES are clonal hematopoietic stem cell disorders highly heterogeneous. This disease is characterized by bone marrow failure that led to peripheral blood citopenias. The outcome of these patients is closely related to cytopenias and to an increased risk to acute myeloid leukemia (AML) progression. Nowadays, these patients are classified according to the revised version (2008) of the WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, which includes 7 subcategories. Due to high heterogeneity, the well-known scoring system IPSS-R (International Prognostic Scoring System) have been developed to better assess the overall survival (OS) their risk to progress. IPSS-R includes 3 variables, cytopenias at peripheral blood, bone marrow blast percentage and karyotype. The last one has shown a high influence in the IPSS-R. We hypothesize that alterations detected by molecular techniques could help us to better define patients with MDS. To elucidate this hypothesis, we studied three groups of MDS patients with different karyotypic alterations, patients with MDS and monosomal karyotype (MK), patients with alterations in chromosome 7 by FISH and patients with MDS or AML and del(5q). MK and alterations of chromosome 7 are included in high risk MDS, whether patients with isolated del(5q) are included in low risk MDS. However, when the alteration is detected in a complex karyotype, the outcome is significantly worse. These three group of patients were studied separately. First we studied the effect of MK. We studied a total of 1,054 adult patients with MDS and altered karyotype. Our main objective was to describe the incidence, characteristics and prognosis of patients with MK and its relation with OS and the progression risk to AML. This study helped us to determine that the worse prognosis related to MK (included in a complex karyotype) is karyotype complexity. This is the one that have a negative impact on patient's prognosis with lower OS and higher risk to AML progression. Second, we studied by FISH a total of 820 patients with MDS without monosomy 7 or 7q deletion by conventional cytogenetics. Our objective was to define the impact of FISH detection (7- or 7q-) in the outcome of MDS patients. A total of 5.2% of cases were positive by FISH. This is of note because -7 and 7q- are related with worse outcome and more aggressive treatments. Moreover patients with an intermediate morphologic risk (WHO 2008) would benefit from a FISH study in order to better classify their prognosis. Due to chromosome 7 alterations worse prognosis, it would be highly recommended to apply FISH 7q in patients with chromosome 7 alterations by conventional cytogenetics who doesn't fulfill clonally criteria. Last group studied were del(5q) patients, this alteration is one of the most common in MDS. We collected 228 patients with del(5q) with myeloid neoplasms (MDS and AML) by applying next-generation sequencing techniques. We were able to determine the 6 genes most commonly mutated: TP53, DNMT3A, CSNK1A1, SF3B1, PRPF8 and ASXL1. Both TP53 and DNMT3A were related to worse prognosis. TP53 and DNMT3A had a negative impact on the OS, but only DNMT3A retained its prognostic impact on the multivariate analysis. Although we couldn't define a mutational profile for patients with del(5q), it would be important to perform initial studies to define patients with a better outcome. Especially when CSNK1A1 is mutated for its correlation with a good lenalidomide's treatment response. In contrast, patients with mutations in TP53 should be closely followed for its well-known negative impact on OS and lenalidomide response. Moreover, due to the high negative impact of DNMT3A mutations in our series. Further studies are needed to clarify its impact on del(5q) patients

Targeted deep sequencing improves outcome stratification in chronic myelomonocytic leukemia with low risk cytogenetic features

Clonal cytogenetic abnormalities are found in 20-30% of patients with chronic myelomonocytic leukemia (CMML), while gene mutations are present in >90% of cases. Patients with low risk cytogenetic features account for 80% of CMML cases and often fall into the low risk categories of CMML prognostic scoring systems, but the outcome differs considerably among them. We performed targeted deep sequencing of 83 myeloid-related genes in 56 CMML patients with low risk cytogenetic features or uninformative conventional cytogenetics (CC) at diagnosis, with the aim to identify the genetic characteristics of patients with a more aggressive disease. Targeted sequencing was also performed in a subset of these patients at time of acute myeloid leukemia (AML) transformation. Overall, 98% of patients harbored at least one mutation. Mutations in cell signaling genes were acquired at time of AML progression. Mutations in ASXL1, EZH2 and NRAS correlated with higher risk features and shorter overall survival (OS) and progression free survival (PFS). Patients with SRSF2 mutations associated with poorer OS, while absence of TET2 mutations (TET2wt) was predictive of shorter PFS. A decrease in OS and PFS was observed as the number of adverse risk gene mutations (ASXL1, EZH2, NRAS and SRSF2) increased. On multivariate analyses, CMML-specific scoring system (CPSS) and presence of adverse risk gene mutations remained significant for OS, while CPSS and TET2wt were predictive of PFS. These results confirm that mutation analysis can add prognostic value to patients with CMML and low risk cytogenetic features or uninformative CC

Targeted deep sequencing of CD34+ cells from peripheral blood can reproduce bone marrow molecular profile in myelodysplastic syndromes

Altres ajuts: José Carreras Leukämie-Stiftung, Award/Grant number: project AR 14/34

Targeted deep sequencing improves outcome stratification in chronic myelomonocytic leukemia with low risk cytogenetic features

Clonal cytogenetic abnormalities are found in 20-30% of patients with chronic myelomonocytic leukemia (CMML), while gene mutations are present in >90% of cases. Patients with low risk cytogenetic features account for 80% of CMML cases and often fall into the low risk categories of CMML prognostic scoring systems, but the outcome differs considerably among them. We performed targeted deep sequencing of 83 myeloid-related genes in 56 CMML patients with low risk cytogenetic features or uninformative conventional cytogenetics (CC) at diagnosis, with the aim to identify the genetic characteristics of patients with a more aggressive disease. Targeted sequencing was also performed in a subset of these patients at time of acute myeloid leukemia (AML) transformation. Overall, 98% of patients harbored at least one mutation. Mutations in cell signaling genes were acquired at time of AML progression. Mutations in ASXL1, EZH2 and NRAS correlated with higher risk features and shorter overall survival (OS) and progression free survival (PFS). Patients with SRSF2 mutations associated with poorer OS, while absence of TET2 mutations (TET2 wt) was predictive of shorter PFS. A decrease in OS and PFS was observed as the number of adverse risk gene mutations (ASXL1, EZH2, NRAS and SRSF2) increased. On multivariate analyses, CMML-specific scoring system (CPSS) and presence of adverse risk gene mutations remained significant for OS, while CPSS and TET2wt were predictive of PFS. These results confirm that mutation analysis can add prognostic value to patients with CMML and low risk cytogenetic features or uninformative CC

Estudio de perfiles moleculares en pacientes con síndrome mielodisplásicos

Los SÍNDROMES MIELODISPLÁSICOS (SMD) son una enfermedad heterogénea clonal de células madre hematopoyéticas. Se caracteriza por una medula ósea ineficaz que produce citopenias en sangre periférica. La severidad de estas citopenias y el riesgo incrementado a progresar a una leucemia mieloide aguda determinarán la evolución de la enfermedad y del paciente. Actualmente estos pacientes se clasifican de acuerdo con 7 subcategorías que establece la revisión del 2008 de la clasificación de la WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. La alta heterogeneidad de cada subcategoría ha hecho necesarios sistemas de puntuación, el más utilizado es el IPSS-R (International Prognostic Scoring System) e incluye 3 variables, las citopenias en sangre periférica, el porcentaje de blastos en médula ósea y el cariotipo. Nos permite evaluar la supervivencia global (SG) y la probabilidad de progresión a leucemia mieloide aguda. La hipótesis global del trabajo es que alteraciones detectadas mediante técnicas moleculares permiten mejorar la definición del pronóstico de pacientes con SMD. Para dilucidar la hipótesis general, se han estudiado tres grupos de pacientes con cariotipos distintos: pacientes con SMD y cariotipo monosómico y/o complejo, pacientes con el cromosoma 7 alterado mediante FISH y pacientes con deleción 5q [del(5q)]. Pacientes con cariotipo monosómico y con alteraciones en el cromosoma 7 se incluyen en el grupo de SMD de alto riesgo. En cambio, pacientes con del(5q), cuando esta se detecta de manera aislada, se incluyen en el grupo de SMD de bajo riesgo. La del(5q) en un cariotipo complejo pasa a ser de pronóstico desfavorable. Primero analizamos el efecto del cariotipo monosómico (MK). Estudiamos 1.054 pacientes con SMD y cariotipo alterado con el objetivo de describir la incidencia, características y pronóstico del MK, y su relación con la SG y el riesgo de progresión a LMA. Este estudio ha permitido determinar que el mal pronóstico asociado a los pacientes con MK, en el contexto de un cariotipo complejo, se debe a la complejidad en el cariotipo lo que les confiere un peor pronóstico, es decir peor SG y mayor riesgo de progresión a LMA. Segundo, estudiamos 820 pacientes por FISH con SMD, su cariotipo no mostró la monosomía 7 o la deleción 7q. El objetivo del estudio era determinar si la detección mediante FISH de estas alteraciones afectaba a su clasificación. El 5,2% de los casos fueron positivos, esto es de importante relevancia ya que la -7 o la del(7q) se relacionan con un impacto pronóstico negativo. Este estudio nos permitió detectar diferencias significativas en la SG de pacientes con riesgo morfológico intermedio (WHO 2008) FISH positivos y negativos. Debido a la relación de estas alteraciones del cromosoma 7 y un peor pronóstico, sería altamente recomendable analizar mediante FISH las alteraciones que no cumplan criterios de clonalidad.a El último grupo de pacientes que estudiamos presentaban la del(5q). Estudiamos un total de 228 pacientes con neoplasias mieloides (SMD y LMA) y del(5q). Mediante el estudio por secuenciación masiva del exoma pudimos determinar los 6 genes más mutados en nuestra serie: TP53, DNMT3A, CSNK1A1, SF3B1, PRPF8 y ASXL1. TP53 y DNMT3A presentaron un efecto negativo en la SG de los pacientes, pero sólo DNMT3A, retuvo la influencia independiente en la SG, en el análisis multivariado. El perfil mutacional de pacientes con del(5q) no se ha podido determinar. Estudios iniciales en pacientes con CSNK1A1 mutado, por su relación con una buena respuesta a la lenalidomida. Los pacientes con TP53 deberían ser controlados de manera frecuente, por el impacto negativo en la SG de mutaciones en este gen. Y, por último, mutaciones en DNMT3A deberían estudiarse en más pacientes con del(5q) para esclarecer su relación.MYELODYSPLASTIC SYNDROMES are clonal hematopoietic stem cell disorders highly heterogeneous. This disease is characterized by bone marrow failure that led to peripheral blood citopenias. The outcome of these patients is closely related to cytopenias and to an increased risk to acute myeloid leukemia (AML) progression. Nowadays, these patients are classified according to the revised version (2008) of the WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, which includes 7 subcategories. Due to high heterogeneity, the well-known scoring system IPSS-R (International Prognostic Scoring System) have been developed to better assess the overall survival (OS) their risk to progress. IPSS-R includes 3 variables, cytopenias at peripheral blood, bone marrow blast percentage and karyotype. The last one has shown a high influence in the IPSS-R. We hypothesize that alterations detected by molecular techniques could help us to better define patients with MDS. To elucidate this hypothesis, we studied three groups of MDS patients with different karyotypic alterations, patients with MDS and monosomal karyotype (MK), patients with alterations in chromosome 7 by FISH and patients with MDS or AML and del(5q). MK and alterations of chromosome 7 are included in high risk MDS, whether patients with isolated del(5q) are included in low risk MDS. However, when the alteration is detected in a complex karyotype, the outcome is significantly worse. These three group of patients were studied separately. First we studied the effect of MK. We studied a total of 1,054 adult patients with MDS and altered karyotype. Our main objective was to describe the incidence, characteristics and prognosis of patients with MK and its relation with OS and the progression risk to AML. This study helped us to determine that the worse prognosis related to MK (included in a complex karyotype) is karyotype complexity. This is the one that have a negative impact on patient’s prognosis with lower OS and higher risk to AML progression. Second, we studied by FISH a total of 820 patients with MDS without monosomy 7 or 7q deletion by conventional cytogenetics. Our objective was to define the impact of FISH detection (7- or 7q-) in the outcome of MDS patients. A total of 5.2% of cases were positive by FISH. This is of note because -7 and 7q- are related with worse outcome and more aggressive treatments. Moreover patients with an intermediate morphologic risk (WHO 2008) would benefit from a FISH study in order to better classify their prognosis. Due to chromosome 7 alterations worse prognosis, it would be highly recommended to apply FISH 7q in patients with chromosome 7 alterations by conventional cytogenetics who doesn’t fulfill clonally criteria. Last group studied were del(5q) patients, this alteration is one of the most common in MDS. We collected 228 patients with del(5q) with myeloid neoplasms (MDS and AML) by applying next-generation sequencing techniques. We were able to determine the 6 genes most commonly mutated: TP53, DNMT3A, CSNK1A1, SF3B1, PRPF8 and ASXL1. Both TP53 and DNMT3A were related to worse prognosis. TP53 and DNMT3A had a negative impact on the OS, but only DNMT3A retained its prognostic impact on the multivariate analysis. Although we couldn’t define a mutational profile for patients with del(5q), it would be important to perform initial studies to define patients with a better outcome. Especially when CSNK1A1 is mutated for its correlation with a good lenalidomide’s treatment response. In contrast, patients with mutations in TP53 should be closely followed for its well-known negative impact on OS and lenalidomide response. Moreover, due to the high negative impact of DNMT3A mutations in our series. Further studies are needed to clarify its impact on del(5q) patients

Correlation of myelodysplastic syndromes with i(17)(q10) and TP53 and SETBP1 mutations.

Letter to the Edito