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INTRODUCTION: Mutated RUNX1 is considered a poor prognostic factor and usually is mutually exclusive with NPM1 mutations. Monitoring of molecular markers for minimal residual disease provides a powerful tool to assess remission and guide clinical decisions. METHODS: Newly diagnosed RUNX1-mutated AML patients, designated to intensive chemotherapy-based treatment or nonintensive regimens, were monitored for mutated RUNX1 transcript levels by qPCR with patient-specific primers. Samples were obtained along the treatment course and follow-up. RESULTS: A clear correlation was observed between mutated RUNX1 levels and response to treatment as observed by flow cytometry and STR-based assessment. CONCLUSION: We demonstrate the feasibility of RUNX1-based MRD to correlate with the clinicopathological status of leukemia. We further suggest how RUNX1 qPCR monitoring can influence clinical decision-making and contribute to improved personalized patient care.
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Subunidade alfa 2 de Fator de Ligação ao Core , Leucemia Mieloide Aguda , Humanos , Neoplasia Residual/diagnóstico , Neoplasia Residual/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Citometria de Fluxo , Reação em Cadeia da Polimerase em Tempo Real , Prognóstico , MutaçãoRESUMO
The JAK2V617F mutation that results in a hyper-activation of the JAK2 kinase in the erythropoietin pathway is a molecular marker for myeloproliferative neoplasms. Using allele-specific Real-Time PCR, we detected the mutation in the blood of 17.3% (17/98) of normal donors; the mutant allele burden was, however, very low (<0.01% compared to >1% in polycythemia vera). It was much higher in differentiated blood cells in the peripheral blood than in undifferentiated CD34+ cells. Erythropoietin-stimulated differentiation of normal CD34+ cells in liquid culture increased the mutation frequency by 3.34-fold. When progenitors from 9 normal donors were grown in erythropoietin-stimulated semi-solid cultures, the mutation was found in 8.69% of the colonies, but only in <3% of the JAK2 alleles in each positive colony, suggesting that the mutation occurred only in a few cells per colony. In mouse erythroleukemia cells carrying human JAK2 DNA, wild-type or JAK2V617F, the frequencies of mutations from JAK2 wild-type to JAK2V617F and vice versa increased following erythroid differentiation. These results suggest that the mutation occurs and accumulates during differentiation. We hypothesize that genetic stability, which relies on DNA repair, is efficient in normal hematopoietic stem cells but is downgraded in differentiating cells, rendering them susceptible to mutations, including JAK2V617F.
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Diferenciação Celular/genética , Janus Quinase 2/genética , Taxa de Mutação , Animais , Doadores de Sangue , Células Precursoras Eritroides/citologia , Eritropoetina/farmacologia , Humanos , Camundongos , Mutação , Transtornos MieloproliferativosRESUMO
Quantitative PCR for specific mutation is being increasingly used in Acute Myeloid Leukemia (AML) to assess Measurable Residual Disease (MRD), allowing for more tailored clinical decisions. To date, standardized molecular MRD is limited to typical NPM1 mutations and core binding factor translocations, with clear prognostic and clinical implications. The monitoring of other identified mutations lacks standardization, limiting its use and incorporation in clinical trials. To overcome this problem, we designed a plasmid bearing both the sequence of the mutation of interest and the ABL reference gene. This allows the use of commercial standards for ABL to determine the MRD response in copy number. We provide technical aspects of this approach as well as our experience with 19 patients with atypical NPM1, RUNX1 and IDH1/2 mutations. In all cases, we demonstrate a correlation between response and copy number. We further demonstrate how copy number monitoring can modulate the clinical management. Taken together, we provide proof of concept of a novel yet simple tool, which allows in-house MRD monitoring for identified mutations, with ABL-based commercial standards. This approach would facilitate large multi-center studies assessing the clinical relevance of selected MRD monitoring.
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Multiple myeloma (MM) is characterized by recurrent relapses. Consequently, patients receive multiple therapy lines, including alkylating agents and immune modulators, which have been associated with secondary malignancies such as myelodysplastic syndrome (MDS). Anti-B-cell maturation antigen (BCMA) chimeric antigen receptor T cell (CART) therapy is efficacious in patients with relapsed/refractory (R/R) MM. However, the long-term complications, particularly MDS, are not well understood. Whether CART therapy causes or promotes MDS has not been thoroughly investigated. In this study, we explored the causal relationship between MDS and CART therapy. We retrospectively examined the prevalence of MDS-related morphological and mutational changes before and after administration of CART therapy in five patients. Among them, four developed MDS after CART therapy, while one had pre-existing MDS prior to CART. None of the four patients who developed post-CART MDS showed morphological MDS changes prior to CART therapy. However, all four patients exhibited molecular alterations associated with MDS in their pre-CART as well as post-CART therapy bone marrow. No new mutations were observed. Our findings provide initial evidence suggesting that anti-BCMA CART therapy in MM may promote expansion of pre-existing MDS clones rather than causing development of new clones.
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The JAK2 V617F mutation is responsible for the constitutive activation of the erythropoietin receptor signaling pathway in most cases of polycythemia vera (PV). The mutation has also been described in healthy people. As smoking may result in secondary polycythemia, the goal of this trial was to examine the effect of smoking on the prevalence of the JAK2 mutation and its correlation to erythrocytosis. The study was case-control. Hospitalized smokers (n = 81) and nonsmokers (n = 61) were recruited. Serum was drawn for complete blood count, erythropoietin, ferritin and venous blood gases. JAK2 mutation was analyzed by highly sensitive allele-specific Quantitative Real Time PCR. The JAK2 mutation was found in 29/81 (35.8%) of smokers in comparison to only 9/61 (14.8%) of the control group (P = 0.007). The frequency of the mutation among smokers who were positive for the JAK2 mutation had a mean of 6.78 × 10(-4) ± 1.08 × 10(-3) vs. 1.51 × 10(-4) ± 2.04 × 10(-4) among nonsmokers (P = 0.027). Both frequencies are much lower than those found in PV. There was a medium correlation between older age and mutation frequency in nonsmokers (r= 0.67, P = 0.043). Hematocrit was higher in smokers (47.8 ± 6 vs. 41.7 ± 4.7, P < 0.0001), but no correlation was found to JAK2 mutation. In a cohort of hospitalized smokers and nonsmokers, JAK2 mutation was more prevalent and found in higher frequencies among smokers than nonsmokers. We suggest that accelerated erythropoiesis renders the cells susceptible to JAK2 mutation.
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Janus Quinase 2/genética , Taxa de Mutação , Fumar/genética , Idoso , Códon , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Policitemia/epidemiologia , Policitemia/genética , Prevalência , Fumar/epidemiologiaRESUMO
We retrospectively studied the prognostic role of molecular (gene rearrangement, GRR) bone marrow (BM) involvement in diffuse large B-cell lymphoma (DLBCL, 424 patients) and in peripheral T-cell lymphoma (PTCL, 67 patients). When correlating BM GRR to histological findings at diagnosis, the GRR test was more sensitive (p = 0.036) but less specific (p < 0.0001) in PTCL than in DLBCL. For DLBCL (but not PTCL), a positive BM GRR correlated with advanced stage (p = 0.0001) and high IPI (p = 0.002), and worsened the progression free survival (PFS) (p = 0.05) and overall survival (OS) (p = 0.01), irrespective of rituximab treatment. Histologic negative/GRR positive cases had worse PFS/OS (p < 0.0001) than histologic/GRR double negative cases, however BM GRR was not an independent prognostic survival factor. End-of-treatment BM GRR did not predict survival. We conclude that BM GRR is unjustified as a prognostic tool for PTCL and should be reserved for a subset of DLBCL patients with negative histology of the BM.
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Neoplasias da Medula Óssea/secundário , Linfoma/genética , Linfoma/patologia , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Progressão da Doença , Feminino , Humanos , Linfoma/tratamento farmacológico , Linfoma/mortalidade , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/mortalidade , Linfoma Difuso de Grandes Células B/patologia , Linfoma de Células T Periférico/tratamento farmacológico , Linfoma de Células T Periférico/genética , Linfoma de Células T Periférico/mortalidade , Linfoma de Células T Periférico/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Estudos Retrospectivos , Análise de Sobrevida , Resultado do TratamentoRESUMO
Many immune functions decline with age and may jeopardize the elderly, as illustrated, for example by the significantly higher mortality rate from influenza in old age. Although innate and humoral immunity are affected by aging, it is the T cell compartment, which manifests most alterations. The mechanisms behind these alterations are still unclear, and several explanations have been offered including thymic involution and Telomere attrition leading to cell senescence. Age related accumulation of mutations has been documented and could serve as an additional mechanism of T cell dysfunction. One effective repair mechanism capable of rectifying errors in DNA replications is the mismatch repair (MMR) system. We previously reported a comparative examination of individual DNA samples from blood cells obtained at 10 year intervals from young and old subjects. We showed significantly higher rates of microsatellite instability (MSI), an indicator of MMR dysfunction in older subjects, compared to young. In the present study we confirm this result, using direct automated sequencing and in addition, we demonstrate that as CD8 lymphocytes from aged individuals, undergo repeated population doublings (PDs) in culture, they develop MSI. CD4 clones that also undergo repeated PDs in culture develop significant MSI as well. Elucidation of this previously unexplored facet of lymphocyte dynamics in relation to aging may help identify novel mechanisms of immunosenescence and pathways that could serve as targets for interventions to restore immune function.
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Envelhecimento/genética , Envelhecimento/imunologia , Repetições de Microssatélites/genética , Linfócitos T/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Pareamento Incorreto de Bases/genética , Pareamento Incorreto de Bases/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/patologia , Senescência Celular/genética , Senescência Celular/imunologia , Metilação de DNA , Reparo do DNA/genética , Reparo do DNA/imunologia , Humanos , Repetições de Microssatélites/imunologia , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/genéticaRESUMO
Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) have traditionally been considered as two distinct entities. However, there are rare reports of patients that, over time, develop both diseases. It remains unresolved whether the origin of the two diseases is from the same clone. In this study, we attempted to retrospectively investigate the clinical and molecular aspects of patients who developed both lymphomas. The rearranged immunoglobulin heavy-chain variable region genes from both diagnoses were compared to each other. Twenty-six patients presented with both diagnoses. Twelve had HL as the primary disorder ("HL first" group) and the majority of these (75%) presented with aggressive lymphoma as the second lymphoma. In contrast, in the 11 patients for whom NHL was the primary disorder ("NHL first" group), this was usually (82%) of low-grade histology. Three patients were diagnosed concurrently with both diseases. Mean age at first diagnosis was higher (p = 0.037) in the NHL first group (56.1 years) than in the HL first group (40 years). Mean time between diagnoses was longer (p = 0.026) in the HL first group (9 years) than in the NHL first group (5 years). For 11 patients, diagnostic samples were available for molecular analyses from both diagnoses of HL and NHL. In 6 of these 11 patients, gene rearrangement studies were informative. No patient had the same gene rearrangement identified in both diseases. It seems that development of HL and NHL in one patient, at different time points, reflects, in many cases, separate biologic diseases.
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Células Clonais/patologia , Doença de Hodgkin/diagnóstico , Linfoma não Hodgkin/diagnóstico , Adulto , Idoso , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Células Clonais/imunologia , Células Clonais/metabolismo , DNA de Neoplasias/análise , DNA de Neoplasias/genética , Chaperona BiP do Retículo Endoplasmático , Feminino , Rearranjo Gênico , Proteínas de Choque Térmico/genética , Doença de Hodgkin/genética , Doença de Hodgkin/imunologia , Humanos , Região Variável de Imunoglobulina/genética , Linfoma não Hodgkin/genética , Linfoma não Hodgkin/imunologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Estudos Retrospectivos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/patologia , Adulto JovemRESUMO
Chronic myelogenous leukaemia (CML) is induced by the Bcr-Abl fusion protein. Inhibition of Bcr-Abl by STI571 is widely used to treat CML patients. Unlike in most cancer types, the frequency of p53 mutations in CML is low. Here, we investigated the effect of STI571 treatment of CML cells on p53 regulation. Exposure of CML cells, including established cell lines and freshly isolated cells from patients, to STI571 reduced p53 protein levels, and severely impaired its accumulation in response to DNA damage. This may be explained by the status of p53 serine 20 phosphorylation. In non-stressed CML cells, serine 20 of p53 is constitutively phosphorylated by Chk1, and is inhibited by STI571. In response to DNA damage, however, this phosphorylation is mediated by Chk1 and Chk2, and is only partially inhibited by STI571. CML cells expressing wild-type p53 are more resistant to treatment with STI571, but moderately more sensitive to DNA damage, than CML cells lacking p53. An enhanced induction of apoptosis by STI571 and DNA damage is observed in CML cells bearing wild-type p53, but not in cells lacking functional p53. This implies that the status of p53 may affect the response of CML cells to this combined treatment.