RESUMEN
We report on a study of next-generation sequencing in 257 patients undergoing investigations for cytopenias. We sequenced bone marrow aspirates using a target enrichment panel comprising 82 genes and used T cells from paired blood as a control. One hundred and sixty patients had idiopathic cytopenias, 81 had myeloid malignancies and 16 had lymphoid malignancies or other diagnoses. Forty-seven of the 160 patients with idiopathic cytopenias had evidence of somatic pathogenic variants consistent with clonal cytopenias. Only 39 genes of the 82 tested were mutated in the 241 patients with either idiopathic cytopenias or myeloid neoplasms. We confirm that T cells can be used as a control to distinguish between germline and somatic variants. The use of paired analysis with a T-cell control significantly reduced the time molecular scientists spent reporting compared to unpaired analysis. We identified somatic variants of uncertain significance (VUS) in a higher proportion (24%) of patients with myeloid malignancies or clonal cytopenias compared to less than 2% of patients with non-clonal cytopenias. This suggests that somatic VUS are indicators of a clonal process. Lastly, we show that blood depleted of lymphocytes can be used in place of bone marrow as a source of material for sequencing.
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Citopenia , Síndromes Mielodisplásicos , Trastornos Mieloproliferativos , Neoplasias , Humanos , Síndromes Mielodisplásicos/genética , Mutación , Linfocitos T/patología , Trastornos Mieloproliferativos/genéticaRESUMEN
The chronic phase of chronic myeloid leukemia (CP-CML) is characterized by the excessive production of maturating myeloid cells. As CML stem/progenitor cells (LSPCs) are poised to cycle and differentiate, LSPCs must balance conservation and differentiation to avoid exhaustion, similar to normal hematopoiesis under stress. Since BCR-ABL1 tyrosine kinase inhibitors (TKIs) eliminate differentiating cells but spare BCR-ABL1-independent LSPCs, understanding the mechanisms that regulate LSPC differentiation may inform strategies to eliminate LSPCs. Upon performing a meta-analysis of published CML transcriptomes, we discovered that low expression of the MS4A3 transmembrane protein is a universal characteristic of LSPC quiescence, BCR-ABL1 independence, and transformation to blast phase (BP). Several mechanisms are involved in suppressing MS4A3, including aberrant methylation and a MECOM-C/EBPε axis. Contrary to previous reports, we find that MS4A3 does not function as a G1/S phase inhibitor but promotes endocytosis of common ß-chain (ßc) cytokine receptors upon GM-CSF/IL-3 stimulation, enhancing downstream signaling and cellular differentiation. This suggests that LSPCs downregulate MS4A3 to evade ßc cytokine-induced differentiation and maintain a more primitive, TKI-insensitive state. Accordingly, knockdown (KD) or deletion of MS4A3/Ms4a3 promotes TKI resistance and survival of CML cells ex vivo and enhances leukemogenesis in vivo, while targeted delivery of exogenous MS4A3 protein promotes differentiation. These data support a model in which MS4A3 governs response to differentiating myeloid cytokines, providing a unifying mechanism for the differentiation block characteristic of CML quiescence and BP-CML. Promoting MS4A3 reexpression or delivery of ectopic MS4A3 may help eliminate LSPCs in vivo.
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Proteínas de Ciclo Celular/metabolismo , Endocitosis , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Proteínas de la Membrana/metabolismo , Receptores de Citocinas/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Regulación hacia Abajo , Regulación Leucémica de la Expresión Génica , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Proteínas de la Membrana/genética , Ratones , Transcriptoma , Células Tumorales CultivadasRESUMEN
The implementation of whole genome sequencing and large somatic gene panels in haematological malignancies is identifying an increasing number of individuals with either potential or confirmed germline predisposition to haematological malignancy. There are currently no national or international best practice guidelines with respect to management of carriers of such variants or of their at-risk relatives. To address this gap, the UK Cancer Genetics Group (UKCGG), CanGene-CanVar and the NHS England Haematological Oncology Working Group held a workshop over two days on 28-29th April 2022, with the aim of establishing consensus guidelines on relevant clinical and laboratory pathways. The workshop focussed on the management of disease-causing germline variation in the following genes: DDX41, CEBPA, RUNX1, ANKRD26, ETV6, GATA2. Using a pre-workshop survey followed by structured discussion and in-meeting polling, we achieved consensus for UK best practice in several areas. In particular, high consensus was achieved on issues regarding standardised reporting, variant classification, multidisciplinary team working and patient support. The best practice recommendations from this meeting may be applicable to an expanding number of other genes in this setting.
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Predisposición Genética a la Enfermedad , Neoplasias Hematológicas , Humanos , Medicina Estatal , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/terapia , Mutación de Línea Germinal , Inglaterra , Células GerminativasRESUMEN
The clinical outcome of chronic myeloid leukaemia patients has vastly improved since the introduction of tyrosine kinase inhibitor treatment, with a significant proportion of patients able to achieve treatment-free remission. However, studies have shown that patients with the e13a2 transcript were less likely to achieve major molecular response compared to those with e14a2 transcripts. Most quantitative polymerase chain reaction (PCR) assays for detection of the BCR-ABL1 fusion gene do not differentiate between the two transcripts and we therefore hypothesised that technical bias linked to the qPCR assay could partially explain the discrepancy in outcomes. We designed an e14a2-specific assay and identified no difference in results compared to an e13a2 standard assay. We then demonstrated that the commercial e14a2 standards were causing a significant overestimation of the e13a2 transcripts. Finally, we reviewed patient management after the qPCR values were corrected, using our new evaluation. We concluded that despite statistically significant differences in qPCR results, there was no impact on patient management or outcome. We conclude that, at least in our institution, it would be inappropriate to perform separate assays for patients with e13a2 or e14a2.
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Leucemia Mielógena Crónica BCR-ABL Positiva , Proteínas de Fusión bcr-abl/genética , Humanos , Mesilato de Imatinib/uso terapéutico , Leucemia Mielógena Crónica BCR-ABL Positiva/diagnóstico , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , TecnologíaRESUMEN
Background: KRAS mutations are present in up to 30% of patients with lung adenocarcinoma. The two most common KRAS mutations in non-small cell lung cancer (NSCLC) are G12C (~40%) and G12V (~22%). We describe the case of a 63-year-old Asian male patient with a very aggressive lung adenocarcinoma harbouring two coexisting missense mutations in the same exon. Methods: The patient presented with a 6 cm spiculated lung mass and bilateral mediastinal lymphadenopathy on imaging. A cytology sample was obtained from EBUS-TBNA of mediastinal lymph nodes, and mutation screening was performed by next-generation sequencing using the Ion Torrent Cancer Hotspot panel. Results: Cytological examination and immunocytochemistry confirmed the presence of metastatic lung adenocarcinoma. The molecular analysis revealed the coexistence of two missense mutations: c.34G > T; p.(Gly12Cys) and c.38G > T; A; p.(Gly13Asp) in exon 2 of the KRAS gene. The two independent variants were confirmed on Integrative Genomic Viewer (IGV), suggesting molecularly independent clones. The patient was treated with palliative care and died within two months of the diagnosis. Conclusions: The present case showed aggressive clinical behaviour. It is questionable whether this aggressive course was due to the coexistence of multiple mutations or to a specific single mutation. Data in the literature regarding the outcome of polyclonal KRAS polyclonal lung adenocarcinomas are scarce, but some evidence seems to indicate that specific mutations may have prognostic value, possibly depending on the disease setting.
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Adenocarcinoma del Pulmón , Adenocarcinoma , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Adenocarcinoma/diagnóstico , Adenocarcinoma/genética , Adenocarcinoma/patología , Adenocarcinoma del Pulmón/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Humanos , Pulmón/patología , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Mutación Missense , Pronóstico , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas ras/genéticaRESUMEN
Few effective therapies exist for acute myeloid leukaemia (AML), in part due to the molecular heterogeneity of this disease. We sought to identify genes crucial to deregulated AML signal transduction pathways which, if inhibited, could effectively eradicate leukaemia stem cells. Due to difficulties in screening primary cells, most previous studies have performed next-generation sequencing (NGS) library knockdown screens in cell lines. Using carefully considered methods including evaluation at multiple timepoints to ensure equitable gene knockdown, we employed a large NGS short hairpin RNA (shRNA) knockdown screen of nearly 5 000 genes in primary AML cells from six patients to identify genes that are crucial for leukaemic survival. Across various levels of stringency, genome-wide bioinformatic analysis identified a gene in the NOX family, NOX1, to have the most consistent knockdown effectiveness in primary cells (P = 5â39 × 10-5 , Bonferroni-adjusted), impacting leukaemia cell survival as the top-ranked gene for two of the six AML patients and also showing high effectiveness in three of the other four patients. Further investigation of this pathway highlighted NOX2 as the member of the NOX family with clear knockdown efficacy. We conclude that genes in the NOX family are enticing candidates for therapeutic development in AML.
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Leucemia Mieloide Aguda/genética , Descubrimiento de Drogas , Regulación Leucémica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Terapia Genética , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Leucemia Mieloide Aguda/terapia , Terapia Molecular Dirigida , NADPH Oxidasa 2/genéticaRESUMEN
Targeted therapy for chronic myeloid leukaemia (CML) has allowed for a near-normal patient life-expectancy; however, quality of life and aggravation of existing co-morbidities have posed new treatment challenges. In clinical practice, TKI dose reduction occurs frequently, often on multiple occasions, because of intolerance. We conducted a retrospective 'real-world practice' review of 246 patients receiving lower than standard dose (LD) TKI after the achievement of major molecular response (MR3), because of intolerable adverse events. In 274 of 298 cases of dose reduction (91·9%), MR3 was maintained at median follow-up of 27·3 months. One patient progressed to blast crisis while on LD TKI. Two patients developed two new ABL kinase domain mutations (T315I and V299L), of whom one had achieved deep molecular response on an alternative LD TKI at last follow-up. Seventy-six patients eventually discontinued LD TKI and the two-year treatment-free remission (TFR) rate in these patients was 74·1%. The majority of patients with CML in at least MR3 appear to be safely managed with LD TKI, although three of 246 patients had new events (progression and new mutation), indicating that this approach requires vigilance. TKI LD does not prevent the achievement of TFR in this patient population.
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Reducción Gradual de Medicamentos/métodos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Inducción de Remisión/métodos , Adulto , Anciano , Anciano de 80 o más Años , Compuestos de Anilina/administración & dosificación , Compuestos de Anilina/efectos adversos , Compuestos de Anilina/uso terapéutico , Comorbilidad , Dasatinib/administración & dosificación , Dasatinib/efectos adversos , Dasatinib/uso terapéutico , Femenino , Estudios de Seguimiento , Proteínas de Fusión bcr-abl/genética , Humanos , Mesilato de Imatinib/administración & dosificación , Mesilato de Imatinib/efectos adversos , Mesilato de Imatinib/uso terapéutico , Leucemia Mielógena Crónica BCR-ABL Positiva/diagnóstico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Masculino , Persona de Mediana Edad , Mutación , Nitrilos/administración & dosificación , Nitrilos/efectos adversos , Nitrilos/uso terapéutico , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/efectos adversos , Pirimidinas/administración & dosificación , Pirimidinas/efectos adversos , Pirimidinas/uso terapéutico , Calidad de Vida , Quinolinas/administración & dosificación , Quinolinas/efectos adversos , Quinolinas/uso terapéutico , Estudios Retrospectivos , Seguridad , Resultado del TratamientoRESUMEN
BACKGROUND & AIMS: Tumour mutational burden (TMB) predicts improved response and survival to immunotherapy. In this pilot study, we optimized targeted next-generation sequencing (tNGS) to estimate TMB in hepatocellular carcinoma (HCC). METHODS: We sequenced 48 non-paired samples (21 fresh-frozen [FF] and 27 paraffin-embedded [FFPE]), among which 11 FFPE samples were pretreated with uracil-DNA glycosylase (UDG). Thirty samples satisfied post-sequencing quality control. High/low TMB was defined by median number of mutations/Mb (Mut/Mb), across different minimum allele frequency (MAF) thresholds (≥0.05, ≥0.1 and ≥0.2). RESULTS: Eligible patients (n = 29) were cirrhotic (84%) with TNM stage I-II HCC (75%). FFPE samples had higher TMB (median 958.39 vs 2.51 Mut/Mb, P < .0001), estimated deamination counts (median 1335.50 vs 0, P < .0001) and C > T transitions at CpG sites (median 60.3% vs 9.1%, P = .002) compared to FF. UDG-treated samples had lower TMB (median 4019.92 vs 353 Mut/Mb, P = .041) and deamination counts (median 6393.5 vs 328.5, P = .041) vs untreated FFPE. At 0.2 MAF threshold with UDG treatment, median TMB was 5.48 (range 1.68-16.07) and did not correlate with salient pathologic features of HCC, including survival. CONCLUSION: While tNGS on fresh HCC samples appears to be the optimal source of tumour DNA, the low median TMB values observed may limit the role of TMB as a predictor of response to immunotherapy in HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Neoplasias Hepáticas/genética , Mutación , Proyectos PilotoRESUMEN
The majority of patients with newly diagnosed chronic myeloid leukemia (CML) will enjoy a life expectancy equivalent to that of unaffected individuals, but will remain on life-long treatment with a concomitant requirement for on-going hospital interactions for molecular monitoring and drug dispensing. In order to determine more accurately the frequency of monitoring required, we performed a 'real-life' retrospective single-center cohort study of 450 patients with CML in at least major molecular remission (MR3) to analyze the risk of loss of MR3 [defined as at least 2 consecutive real-time quantitative polymerase chain reaction (RT-qPCR) results >0.1% International Scale (IS)]. Patients who achieved sustained MR4 (sMR4, BCR-ABL1 RT-qPCR <0.01% IS for 12 months) had a probability of loss of MR3 at 1 and 5 years of 0 and 2.6% (95%CI: 1.2-5.4) respectively, compared to 4.4% (95%CI: 1.9-9.8) and 25.4% (95%CI: 16.7-36.7) respectively, in those who achieved sustained MR3 (sMR3) but not sMR4 (P<0.001). No patient who improved their response to a deep molecular level (at least MR4) lost MR3 if they were considered compliant, had no history of resistance and remained on standard dose tyrosine kinase inhibitor (TKI). MR4 maintained for at least one year represents a secure response threshold for patients with CML, after which no MR3 loss occurs if certain conditions are satisfied (standard TKI dose, full compliance, and lack of previous TKI resistance). This finding may justify reduction of the frequency of hospital interaction, with an associated positive impact on quality of life, survivorship, and economic burden to both patients and healthcare providers.
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Proteínas de Fusión bcr-abl/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/diagnóstico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Adulto , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor , Femenino , Humanos , Estimación de Kaplan-Meier , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/mortalidad , Masculino , Persona de Mediana Edad , Pronóstico , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/efectos adversos , Inhibidores de Proteínas Quinasas/uso terapéutico , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Transcripción Genética , Adulto JovenRESUMEN
There are no validated molecular biomarkers to identify newly-diagnosed individuals with chronic-phase chronic myeloid leukemia likely to respond poorly to imatinib and who might benefit from first-line treatment with a more potent second-generation tyrosine kinase inhibitor. Our inability to predict these 'high-risk' individuals reflects the poorly understood heterogeneity of the disease. To investigate the potential of genetic variants in epigenetic modifiers as biomarkers at diagnosis, we used Ion Torrent next-generation sequencing of 71 candidate genes for predicting response to tyrosine kinase inhibitors and probability of disease progression. A total of 124 subjects with newly-diagnosed chronic-phase chronic myeloid leukemia began with imatinib (n=62) or second-generation tyrosine kinase inhibitors (n=62) and were classified as responders or non-responders based on the BCRABL1 transcript levels within the first year and the European LeukemiaNet criteria for failure. Somatic variants affecting 21 genes (e.g. ASXL1, IKZF1, DNMT3A, CREBBP) were detected in 30% of subjects, most of whom were non-responders (41% non-responders, 18% responders to imatinib, 38% non-responders, 25% responders to second-generation tyrosine kinase inhibitors). The presence of variants predicted the rate of achieving a major molecular response, event-free survival, progression-free survival and chronic myeloid leukemia-related survival in the imatinib but not the second-generation tyrosine kinase inhibitors cohort. Rare germline variants had no prognostic significance irrespective of treatment while some pre-leukemia variants suggest a multi-step development of chronic myeloid leukemia. Our data suggest that identification of somatic variants at diagnosis facilitates stratification into imatinib responders/non-responders, thereby allowing earlier use of second-generation tyrosine kinase inhibitors, which, in turn, may overcome the negative impact of such variants on disease progression.
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Biomarcadores de Tumor/genética , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Mesilato de Imatinib/uso terapéutico , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Mutación , Inhibidores de Proteínas Quinasas/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Estudios de Seguimiento , Perfilación de la Expresión Génica , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Tasa de Supervivencia , Insuficiencia del Tratamiento , Adulto JovenRESUMEN
The mechanisms underlying tyrosine kinase inhibitor (TKI) resistance in chronic myeloid leukemia (CML) patients lacking explanatory BCR-ABL1 kinase domain mutations are incompletely understood. To identify mechanisms of TKI resistance that are independent of BCR-ABL1 kinase activity, we introduced a lentiviral short hairpin RNA (shRNA) library targeting â¼5000 cell signaling genes into K562(R), a CML cell line with BCR-ABL1 kinase-independent TKI resistance expressing exclusively native BCR-ABL1. A customized algorithm identified genes whose shRNA-mediated knockdown markedly impaired growth of K562(R) cells compared with TKI-sensitive controls. Among the top candidates were 2 components of the nucleocytoplasmic transport complex, RAN and XPO1 (CRM1). shRNA-mediated RAN inhibition or treatment of cells with the XPO1 inhibitor, KPT-330 (Selinexor), increased the imatinib sensitivity of CML cell lines with kinase-independent TKI resistance. Inhibition of either RAN or XPO1 impaired colony formation of CD34(+) cells from newly diagnosed and TKI-resistant CML patients in the presence of imatinib, without effects on CD34(+) cells from normal cord blood or from a patient harboring the BCR-ABL1(T315I) mutant. These data implicate RAN in BCR-ABL1 kinase-independent imatinib resistance and show that shRNA library screens are useful to identify alternative pathways critical to drug resistance in CML.
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Transporte Activo de Núcleo Celular , Proteínas de Fusión bcr-abl/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , ARN Interferente Pequeño/genética , Transporte Activo de Núcleo Celular/genética , Benzamidas/farmacología , Línea Celular Tumoral , Supervivencia Celular , Resistencia a Antineoplásicos/genética , Proteínas de Fusión bcr-abl/genética , Técnicas de Silenciamiento del Gen , Biblioteca de Genes , Humanos , Hidrazinas/farmacología , Mesilato de Imatinib , Células K562 , Carioferinas/antagonistas & inhibidores , Carioferinas/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Mutación , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/genética , Transducción de Señal , Triazoles/farmacología , Ensayo de Tumor de Célula Madre , Proteína de Unión al GTP ran/antagonistas & inhibidores , Proteína de Unión al GTP ran/genética , Proteína Exportina 1Asunto(s)
Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/patología , Células Madre Mesenquimatosas/citología , Células Madre Neoplásicas/citología , Anciano , Células Cultivadas , Técnicas de Cocultivo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/terapia , Células Madre Mesenquimatosas/metabolismo , Persona de Mediana Edad , Terapia Molecular Dirigida , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Células Madre Neoplásicas/metabolismo , Transducción de Señal/genética , Transcriptoma , Células Tumorales Cultivadas , Microambiente Tumoral , Adulto JovenAsunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Trasplante de Médula Ósea , Resistencia a Antineoplásicos , Leucemia Mielógena Crónica BCR-ABL Positiva , Adolescente , Adulto , Aloinjertos , Compuestos de Anilina/uso terapéutico , Niño , Preescolar , Dasatinib/uso terapéutico , Femenino , Hematología , Humanos , Mesilato de Imatinib/uso terapéutico , Imidazoles/uso terapéutico , Interferón-alfa/uso terapéutico , Leucemia Mielógena Crónica BCR-ABL Positiva/diagnóstico , Leucemia Mielógena Crónica BCR-ABL Positiva/terapia , Masculino , Nitrilos/uso terapéutico , Piridazinas/uso terapéutico , Pirimidinas/uso terapéutico , Quinolinas/uso terapéutico , Sociedades Médicas , Reino UnidoRESUMEN
BCR-ABL1 compound mutations can confer high-level resistance to imatinib and other ABL1 tyrosine kinase inhibitors (TKIs). The third-generation ABL1 TKI ponatinib is effective against BCR-ABL1 point mutants individually, but remains vulnerable to certain BCR-ABL1 compound mutants. To determine the frequency of compound mutations among chronic myeloid leukemia patients on ABL1 TKI therapy, in the present study, we examined a collection of patient samples (N = 47) with clear evidence of 2 BCR-ABL1 kinase domain mutations by direct sequencing. Using a cloning and sequencing method, we found that 70% (33/47) of double mutations detected by direct sequencing were compound mutations. Sequential, branching, and parallel routes to compound mutations were common. In addition, our approach revealed individual and compound mutations not detectable by direct sequencing. The frequency of clones harboring compound mutations with more than 2 missense mutations was low (10%), whereas the likelihood of silent mutations increased disproportionately with the total number of mutations per clone, suggesting a limited tolerance for BCR-ABL1 kinase domain missense mutations. We conclude that compound mutations are common in patients with sequencing evidence for 2 BCR-ABL1 mutations and frequently reflect a highly complex clonal network, the evolution of which may be limited by the negative impact of missense mutations on kinase function.
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Proteínas de Fusión bcr-abl/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Mutación Missense/genética , Piperazinas/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/uso terapéutico , Benzamidas , Clonación Molecular , Análisis Mutacional de ADN/métodos , Resistencia a Antineoplásicos/genética , Proteínas de Fusión bcr-abl/química , Regulación Leucémica de la Expresión Génica/genética , Humanos , Mesilato de Imatinib , Estructura Terciaria de ProteínaAsunto(s)
Leucemia Mielógena Crónica BCR-ABL Positiva , Resistencia a Antineoplásicos/genética , Proteínas de Fusión bcr-abl/genética , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
Relapse following a short clinical response to therapy is the major challenge for the management of acute myeloid leukemia (AML) patients. Leukemic stem cells (LSC), as the source of relapse, have been investigated for their metabolic preferences and their alterations at the time of relapse. As LSC rely on oxidative phosphorylation (OXPHOS) for energy requirement, reactive oxygen species (ROS), as by-products of OXPHOS, have been investigated for their role in the effectiveness of the standard AML therapy. Increased levels of non-mitochondrial ROS, generated by nicotinamide adenine dinucleotide phosphate oxidase, in a subgroup of AML patients add to the complexity of studying ROS. Although there are various studies presenting the contribution of ROS to AML pathogenesis, resistance, and its inhibition or activation as a target, a model that can clearly explain its role in AML has not been conceptualized. This is due to the heterogeneity of AML, the dynamics of ROS production, which is influenced by factors such as the type of treatment, cell differentiation state, mitochondrial activity, and also the heterogeneous generation of non-mitochondrial ROS and limited available data on their interaction with the microenvironment. This review summarizes these challenges and the recent progress in this field.
RESUMEN
Second-generation tyrosine kinase inhibitors (2GTKI) are more effective in inducing rapid molecular responses than imatinib when used first-line in patients with chronic myeloid leukemia in chronic phase (CML-CP). However, failure of first line-2GTKI (1L-2GTKI) still occurs and there is no consensus regarding subsequent management. We retrospectively analyzed the outcome of 106 CML-CP patients treated with 1L-2GTKI and with a median follow-up of 91 months. 45 patients (42.4%) switched to an alternative TKI, 28 for intolerance (26.4%) and 17 (16%) for resistance. Most patients who remained on 1L-2GTKI achieved deep molecular responses (DMR) and 15 (14.1%) are in treatment-free remission (TFR). Intolerant patients also obtained DMR, although most required multiple TKI changes and were slower to respond, particularly if treated with 2L-imatinib. Inferior outcomes were observed in resistant patients, who failed alternative 2L-2GTKI and required 3/4GTKI and/or allogeneic hematopoietic stem cell transplant (alloSCT). 7yr-OS was significantly lower for these individuals (66.1%) than for intolerant patients and those who remained on 1L-2GTKI (100% and 97.9%, respectively; p = 0.001). It is apparent that failure of 1L-2GTKI is a challenging problem in modern CML therapy. Intolerance can be effectively managed by switching to an alternative 2GTKI, but resistance requires early consideration of 3/4GTKI.
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Leucemia Mielógena Crónica BCR-ABL Positiva , Humanos , Mesilato de Imatinib/uso terapéutico , Dasatinib , Inhibidores de Proteínas Quinasas/efectos adversos , Estudios Retrospectivos , Proteínas de Fusión bcr-abl , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológicoRESUMEN
We studied the relation between adherence to imatinib measured with microelectronic monitoring systems and the probabilities of losing a complete cytogenetic response (CCyR) and of imatinib failure in 87 CCyR chronic myeloid leukemia patients receiving long-term therapy. We included in our analysis the most relevant prognostic factors described to date. On multivariate analysis, the adherence rate and having failed to achieve a major molecular response were the only independent predictors for loss of CCyR and discontinuation of imatinib therapy. The 23 patients with an adherence rate less than or equal to 85% had a higher probability of losing their CCyR at 2 years (26.8% vs 1.5%, P = .0002) and a lower probability of remaining on imatinib (64.5% vs 90.6%, P = .006) than the 64 patients with an adherence rate more than 85%. In summary, we have shown that poor adherence is the principal factor contributing to the loss of cytogenetic responses and treatment failure in patients on long-term therapy.
Asunto(s)
Aberraciones Cromosómicas , Resistencia a Antineoplásicos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Cooperación del Paciente , Piperazinas/uso terapéutico , Pirimidinas/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/uso terapéutico , Benzamidas , Aberraciones Cromosómicas/efectos de los fármacos , Aberraciones Cromosómicas/estadística & datos numéricos , Supervivencia sin Enfermedad , Femenino , Estudios de Seguimiento , Humanos , Mesilato de Imatinib , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Masculino , Persona de Mediana Edad , Cooperación del Paciente/estadística & datos numéricos , Inducción de Remisión , Factores de Tiempo , Insuficiencia del TratamientoRESUMEN
BACKGROUND: Chronic myeloid leukaemia (CML) is one of the most well characterised human malignancies. Most patients have a cytogenetically visible translocation between chromosomes 9 and 22 which generates the pathognomonic BCR::ABL1 fusion gene. The derivative chromosome 22 ('Philadelphia' or Ph chromosome) usually harbours the fusion gene encoding a constitutively active ABL1 kinase domain. A small subset of patients have no visible translocation. Historically, these 'Philadelphia chromosome negative' patients caused diagnostic confusion between CML and other myeloproliferative neoplasms; it is now well established that the BCR::ABL1 fusion gene can be generated via submicroscopic intrachromosomal insertion of ABL1 sequence into BCR, or, more rarely, of BCR into ABL1. The fusion genes arising from cryptic insertions are not detectable via G-banded chromosome analysis [karyotype] but can nevertheless always be detected using fluorescence in situ hybridisation (FISH) and/or qualitative reverse transcriptase PCR. CASE PRESENTATION: A 43-year-old female presented with suspected CML in 2007; however, contemporaneous gold standard laboratory investigations, G-banded chromosome analysis and FISH, were both negative. The reverse transcriptase quantitative PCR (RT-qPCR) assay available at the time, which was capable of detecting the common BCR::ABL1 transcripts (e13a2/e14a2), was also negative. Upon review in 2009, the newly recommended reverse transcriptase multiplex PCR (capable of detecting all BCR::ABL1 transcripts including the atypical ones) subsequently detected an e19a2 fusion. The patient then responded to tyrosine kinase inhibitor therapy. In contrast, FISH studies of both samples with three commercially available probes remained consistently negative. Retrospective whole genome sequencing, undertaken as part of the 100,000 Genomes Project, has now revealed that the patient's BCR::ABL1 fusion gene arose via a uniquely small insertion of 122 kb ABL1 sequences into BCR. CONCLUSIONS: We present a patient with suspected chronic myeloid leukaemia whose genetic investigations were originally negative at the time of diagnosis despite the use of contemporaneous gold standard methods. This is the first report of a FISH-negative, BCR::ABL1 positive CML which demonstrates that, even after sixty years of research into one of the most well understood human malignancies, whole genome sequencing can yield novel diagnostic findings in CML.