Estudio del gen IKZF1 y de microdeleciones intragénicas en la leucemia linfoblástica aguda

La leucemia Linfoblástica Aguda (LLA) es una neoplasia caracterizada por una gran diversidad de alteraciones genéticas. En los últimos años, se ha descrito la ocurrencia de pequeñas deleciones focales en genes de relevancia para la célula linfoide. En concreto, las que afectan a IKZF1 parecen tener valor pronóstico en la edad pediátrica. OBJETIVOS: Estudiar la presencia de microdeleciones en los genes CDKN2A, CDKN2B, PAX5, ETV6, EBF1, BTG1, RB1, la región pseudoautosómica del cromosoma X/Y (PAR1) en una serie de pacientes con LLA. Además, estudiar la presencia de mutaciones, microdeleciones y alteración del patrón de expresión de isoformas del gen IKZF1 en estos pacientes. MÉTODOS: Se reunió una serie de 342 pacientes (164 niños y 178 adultos) de 0 a 85 años. El 85% mostró fenotipo B y el resto T. El estudio de microdeleciones se realizó mediante MLPA (MRC Holland) con el kit P0335 que incluyó sondas para todos los genes de interés. El estudio de mutaciones se realizó en todos los exones codificantes del gen IKZF1 mediante técnica de cribado por High Resolution Melting y posterior confirmación con secuenciación tipo Sanger. El estudio del patrón de isoformas se realizó mediante RT-PCR y lectura en gel de agarosa. RESULTADOS: Hallamos alteraciones del gen IKZF1 en el 26% de los pacientes. Sólo se hallaron en la LLA de estirpe B, su frecuencia aumentó progresivamente con la edad y ocurrieron con mayor frecuencia en la LLA-Ph positiva (71%) que en el resto (20%). Casi la totalidad (97%) de las alteraciones de IKZF1 fueron microdeleciones intragénicas, siendo mutaciones las restantes alteraciones encontradas. La microdeleción más frecuente fue la que afectó a los exones del 4 al 7, resultando en la expresión de la isoforma patológica Ik6. En el resto de casos con microdeleciones observamos ausencia completa de expresión o un patrón normal de isoformas. Las microdeleciones intragénicas se hallaron con igual frecuencia en la LLA-B (63%) y la LLA-T (62%), tanto pediátricas (62%) como de adulto (63%). Los genes más frecuentemente involucrados fueron CDKN2A (30%), CDKN2B (28%), PAX5 (18%) y ETV6 (10%), que se hallan de manera concomitante en muchos de los casos. Las deleciones de CDKN2A y CDKN2B fueron más frecuentes en las LLA-T, mientras que en las de BTG1 y EBF1 ocurrieron exclusivamente en las LLA-B. En los pacientes pediátricos, la estratificación de riesgo citogenético fue el factor que más influyó en la supervivencia y en la probabilidad de recaída. En los pacientes adultos, las microdeleciones de CDKN2A/B y las de ETV6 mostraron tener valor pronóstico independiente para la supervivencia global, mientras que para la probabilidad de recaída lo han sido las deleciones de ETV6 y la concurrencia de ≥ 4 deleciones. Las alteraciones de IKZF1 se han asociado a un pronóstico adverso, si bien los tratamientos aplicados adaptados al riesgo de recaída pueden haber condicionado la ausencia de significación estadística en el análisis multivariante. CONCLUSIONES: Las alteraciones de IKZF1 y las microdeleciones intragénicas son eventos frecuentes en la LLA. Algunas de ellas han demostrado tener valor pronóstico en nuestra serie.Acute Lymphoblastic Leukemia (ALL) is a neoplasm characterized by a great diversity of genetic abnormalities. Recently, it has been described the occurrence of small focal microdeletions involving important genes for the leukemic cell. IKZF1 deletions seem to be of special interest due to the prognostic value shown in pediatric series. AIMS: To study the presence of microdeletions in the CDKN2A, CDKN2B, PAX5, ETV6, EBF1, BTG1, RB1 genes and the pseudoatosomic region of X/Y (PAR1) in a series of patients diagnosed with ALL. To study the presence of mutations, microdeletions and expression of different isoforms of the IKZF1 gene in that series. MÉTODOS: A total of 342 patients diagnosed with ALL was included in this study (164 children y 178 adults) from 0 to 85 years of age. Eighty-five percent of patients showed B-ALL and 15% T-ALL. Microdeletions were studied by MLPA (MRC Holland) with P0335 kit, which includes several probes for the selected genes. Mutational studies were performed in all coding exons of the IKZF1 gene using High Resolution Melting technique as screening and Sanger sequencing for confirmation. Isoform pattern was studied by RT-PCR and amplified products were visualized in agarose gel. RESULTADOS: IKZF1 gene abnormalities were seen in 26% of patients. They were only seen in B-ALL cases, its frequency increased with age and were more frequently found in Philadelphia positive ALL (71%) than in other ALL cases (20%). Almost all abnormalities were microdeletions (97%), and the remaining abnormalities were mutations. The most frequent microdeletion was the one that involved exons 4 to 7, leading to the expression of the aberrant short isoform Ik6. The remaining cases showed a normal expression pattern of isoforms or lack of expression. Gene microdeletions were seen with the same frequency in B-ALL (63%) as in T-ALL (62%) and in children (62%) as in adults (63%). The most frequently involved genes were CDKN2A (30%), CDKN2B (28%), PAX5 (18%) and ETV6 (10%), which were concurrently deleted in several cases. Deletions in CDKN2A y CDKN2B were more frequent in T-ALL, while BTG1 and EBF1 were found exclusively in B-ALL. In pediatric patients, cytogenetic risk stratification was the only independent variable conditioning survival and relapse in multivariate analyses. In adult patients, CDKN2A/B and ETV6 deletions showed an independent prognostic value for overall survival, and ETV6 deletions and the concurrence of ≥ 4 deletions for relapse-free survival. IKZF1 deletions confered a worse prognosis, however, this association was not independent in multivariate analyses, maybe due to the risk-stratified treatment protocols used in this series. CONCLUSIONS: IKZF1 gene abnormalities and gene microdeletions are frequent events in ALL. Some of them have shown prognostic value in our series

Empleo de nuevas tecnologías en el diagnóstico oncohematológico

En las últimas décadas, gracias al empleo de nuevas tecnologías como los microarrays o la secuenciación masiva, hemos presenciado un notable avance en el conocimiento del origen y la evolución de las neoplasias hematológicas. El presente artículo pretende analizar el papel que desempeñan estas tecnologías en el campo de la investigación oncohematológica y su potencial aplicación en la práctica clínica habitual.Thanks to the use of new technologies such as microarrays or next-generation sequencing, we have witnessed over the last decade a huge improvement in the knowledge of the origin and evolution of hematologic neoplasms. This article analyses the role of these technologies in the hemato-oncology research, as well as its potential application in the routine clinical practice.Medicin

CYP2C8 gene polymorphism and bisphosphonate-related osteonecrosis of the jaw in patients with multiple myeloma

Osteonecrosis of the jaw is an uncommon but potentially serious complication of bisphosphonate therapy in multiple myeloma. Previous studies showed that the presence of one or two minor alleles of the cytochrome P450, subfamily 2C polypeptide 8 gene (CYP2C8) polymorphism rs1934951 was an independent prognostic marker associated with development of osteonecrosis of the jaw in multiple myeloma patients treated with bisphosphonates. The aim of this study was to validate the frequency of SNP rs193451 in 79 patients with multiple myeloma. In 9 (22%) patients developing osteonecrosis of the jaw, a heterozygous genotype was found, in contrast with those who did not develop osteonecrosis of the jaw (n=4, 11%) or healthy individuals (n=6, 13%). We found no differences in the cumulative risk of developing osteonecrosis of the jaw between patients homozygous and heterozygous for the major allele. We were unable to confirm a significant association between this polymorphism and the risk of developing osteonecrosis of the jaw

The modular network structure of the mutational landscape of Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is associated with the sequential accumulation of acquired genetic alterations. Although at diagnosis cytogenetic alterations are frequent in AML, roughly 50% of patients present an apparently normal karyotype (NK), leading to a highly heterogeneous prognosis. Due to this significant heterogeneity, it has been suggested that different molecular mechanisms may trigger the disease with diverse prognostic implications. We performed whole-exome sequencing (WES) of tumor-normal matched samples of de novo AML-NK patients lacking mutations in NPM1, CEBPA or FLT3-ITD to identify new gene mutations with potential prognostic and therapeutic relevance to patients with AML. Novel candidate-genes, together with others previously described, were targeted resequenced in an independent cohort of 100 de novo AML patients classified in the cytogenetic intermediate-risk (IR) category. A mean of 4.89 mutations per sample were detected in 73 genes, 35 of which were mutated in more than one patient. After a network enrichment analysis, we defined a single in silico model and established a set of seed-genes that may trigger leukemogenesis in patients with normal karyotype. The high heterogeneity of gene mutations observed in AML patients suggested that a specific alteration could not be as essential as the interaction of deregulated pathways

Real-world effectiveness of caplacizumab vs the standard of care in immune thrombotic thrombocytopenic purpura

Immune thrombotic thrombocytopenic purpura (iTTP) is a thrombotic microangiopathy caused by anti-ADAMTS13 antibodies. Caplacizumab is approved for adults with an acute episode of iTTP in conjunction with plasma exchange (PEX) and immunosuppression. The objective of this study was to analyze and compare the safety and efficacy of caplacizumab vs the standard of care and assess the effect of the concomitant use of rituximab. A retrospective study from the Spanish TTP Registry of patients treated with caplacizumab vs those who did not receive it was conducted. A total of 155 patients with iTTP (77 caplacizumab, 78 no caplacizumab) were included. Patients initially treated with caplacizumab had fewer exacerbations (4.5% vs 20.5%; P <.05) and less refractoriness (4.5% vs 14.1%; P <.05) than those who were not treated. Time to clinical response was shorter when caplacizumab was used as initial treatment vs caplacizumab used after refractoriness or exacerbation. The multivariate analysis showed that its use in the first 3 days after PEX was associated with a lower number of PEX (odds ratio, 7.5; CI, 2.3-12.7; P <.05) and days of hospitalization (odds ratio, 11.2; CI, 5.6-16.9; P <.001) compared with standard therapy. There was no difference in time to clinical remission in patients treated with caplacizumab compared with the use of rituximab. No severe adverse event was described in the caplacizumab group. In summary, caplacizumab reduced exacerbations and refractoriness compared with standard of care regimens. When administered within the first 3 days after PEX, it also provided a faster clinical response, reducing hospitalization time and the need for PEX

Real-world effectiveness of caplacizumab vs the standard of care in immune thrombotic thrombocytopenic purpura

Immune thrombotic thrombocytopenic purpura (iTTP) is a thrombotic microangiopathy caused by anti-ADAMTS13 antibodies. Caplacizumab is approved for adults with an acute episode of iTTP in conjunction with plasma exchange (PEX) and immunosuppression. The objective of this study was to analyze and compare the safety and efficacy of caplacizumab vs the standard of care and assess the effect of the concomitant use of rituximab. A retrospective study from the Spanish TTP Registry of patients treated with caplacizumab vs those who did not receive it was conducted. A total of 155 patients with iTTP (77 caplacizumab, 78 no caplacizumab) were included. Patients initially treated with caplacizumab had fewer exacerbations (4.5% vs 20.5%; P < .05) and less refractoriness (4.5% vs 14.1%; P < .05) than those who were not treated. Time to clinical response was shorter when caplacizumab was used as initial treatment vs caplacizumab used after refractoriness or exacerbation. The multivariate analysis showed that its use in the first 3 days after PEX was associated with a lower number of PEX (odds ratio, 7.5; CI, 2.3-12.7; P < .05) and days of hospitalization (odds ratio, 11.2; CI, 5.6-16.9; P < .001) compared with standard therapy. There was no difference in time to clinical remission in patients treated with caplacizumab compared with the use of rituximab. No severe adverse event was described in the caplacizumab group. In summary, caplacizumab reduced exacerbations and refractoriness compared with standard of care regimens. When administered within the first 3 days after PEX, it also provided a faster clinical response, reducing hospitalization time and the need for PEX

Empleo de nuevas tecnologías en el diagnóstico oncohematológico

Thanks to the use of new technologies such as microarrays or next-generation sequencing, we have witnessed over the last decade a huge improvement in the knowledge of the origin and evolution of hematologic neoplasms. This article analyses the role of these technologies in the hemato-oncology research, as well as its potential application in the routine clinical practice.En las últimas décadas, gracias al empleo de nuevas tecnologías como los microarrays o la secuenciación masiva, hemos presenciado un notable avance en el conocimiento del origen y la evolución de las neoplasias hematológicas. El presente artículo pretende analizar el papel que desempeñan estas tecnologías en el campo de la investigación oncohematológica y su potencial aplicación en la práctica clínica habitual

The Mutational Landscape of Acute Promyelocytic Leukemia Reveals an Interacting Network of Co-Occurrences and Recurrent Mutations.

Preliminary Acute Promyelocytic Leukemia (APL) whole exome sequencing (WES) studies have identified a huge number of somatic mutations affecting more than a hundred different genes mainly in a non-recurrent manner, suggesting that APL is a heterogeneous disease with secondary relevant changes not yet defined. To extend our knowledge of subtle genetic alterations involved in APL that might cooperate with PML/RARA in the leukemogenic process, we performed a comprehensive analysis of somatic mutations in APL combining WES with sequencing of a custom panel of targeted genes by next-generation sequencing. To select a reduced subset of high confidence candidate driver genes, further in silico analysis were carried out. After prioritization and network analysis we found recurrent deleterious mutations in 8 individual genes (STAG2, U2AF1, SMC1A, USP9X, IKZF1, LYN, MYCBP2 and PTPN11) with a strong potential of being involved in APL pathogenesis. Our network analysis of multiple mutations provides a reliable approach to prioritize genes for additional analysis, improving our knowledge of the leukemogenesis interactome. Additionally, we have defined a functional module in the interactome of APL. The hypothesis is that the number, or the specific combinations, of mutations harbored in each patient might not be as important as the disturbance caused in biological key functions, triggered by several not necessarily recurrent mutations