RESUMEN
Longitudinal molecular measurable residual disease (MRD) sampling after completion of therapy serves as a refined tool for identification of imminent relapse of acute myeloid leukaemia (AML) among patients in long-term haematological complete remission. Tracking of increasing quantitative polymerase chain reaction MRD before cytomorphological reappearance of blasts may instigate individual management decisions and has paved the way for development of pre-emptive treatment strategies to substantially delay or perhaps even revert leukaemic regrowth. Traditionally, MRD monitoring is performed using repeated bone marrow aspirations, albeit the current European LeukemiaNet MRD recommendations acknowledge the use of peripheral blood as an alternative source for MRD assessment. Persistent MRD positivity in the bone marrow despite continuous morphological remission is frequent in both core binding factor leukaemias and nucleophosmin 1-mutated AML. In contrast, monthly assessment of MRD in peripheral blood superiorly separates patients with imminent haematological relapse from long-term remitters and may allow pre-emptive therapy of AML relapse.
Asunto(s)
Biomarcadores de Tumor/sangre , Leucemia Mieloide Aguda/sangre , Células Neoplásicas Circulantes , ARN Mensajero/sangre , ARN Neoplásico/sangre , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Médula Ósea/patología , Diagnóstico Precoz , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Neoplasia Residual , Nucleofosmina/genética , Proteínas de Fusión Oncogénica/genética , Valor Predictivo de las Pruebas , ARN Largo no Codificante/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Recurrencia , Inducción de Remisión , Sensibilidad y EspecificidadRESUMEN
Serial assessments of measurable (or minimal) residual disease (MRD) by qPCR may identify nascent relapse in children with acute myeloid leukaemia (AML) and enable pre-emptive therapy. We investigated the kinetics and prognostic impact of recurrent fusion transcripts (RUNX1-RUNX1T1, CBFB-MYH11, KMT2A-MLLT3 or KMT2A-ELL) in 774 post-induction samples from bone marrow (BM, 347) and peripheral blood (PB, 427) from 75 children with AML. BM MRD persistence during consolidation did not increase the risk of relapse, and MRD at therapy completion did not correlate to outcome (HR = 0·64/MRD log reduction (CI: 0·32-1·26), P = 0·19). In contrast, 8/8 patients with detectable MRD in PB after first consolidation relapsed. Persistence (n = 4) and shifting from negative to positive (n = 10) in PB during follow-up predicted relapse in 14/14 patients. All 253 PB samples collected during follow-up from 36 patients in continuous complete remission were MRD negative. In core-binding factor AML, persistent low-level MRD positivity in BM during follow-up was frequent but an increment to above 5 × 10-4 heralded subsequent haematological relapse in 12/12 patients. We demonstrate that MRD monitoring in PB after induction therapy is highly informative and propose an MRD increment above 5 × 10-4 in PB and BM as a definition of molecular relapse since it always leads to haematological relapse.
Asunto(s)
Leucemia Mieloide Aguda/complicaciones , Neoplasia Residual/diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Adolescente , Biomarcadores de Tumor , Niño , Preescolar , Progresión de la Enfermedad , Femenino , Humanos , Lactante , Recién Nacido , Leucemia Mieloide Aguda/sangre , Masculino , Neoplasia Residual/sangreRESUMEN
Preemptive treatment of relapse of acute myeloid leukemia (AML) holds the promise to improve the prognosis of this currently highly lethal condition. Proposed treatment modalities applicable in preemptive cytoreduction (e.g., demethylating agents or standard chemotherapy) differ substantially in interval from administration to antileukemic effect. The t(6;9) balanced translocation, producing the DEK-NUP214 fusion protein, is seen in only 1% of patients with AML. We hypothesized that in these patients, who relapse with a very high frequency, a more detailed knowledge of leukemic relapse growth kinetics would improve the personalized decision-making regarding re-administration of chemotherapy. Based on standardized quantitative PCR data, we therefore delineated the relapse kinetics in a cohort of 27 relapsing DEK-NUP214-positive patients treated in four different European countries. The prerelapse leukemic burden increased with a median doubling time of 13 d (range: 5-51 d, median: 0.71 logs/month, range: 0.18-1.91 logs/month), with FLT3-ITD-positive patients relapsing significantly faster than FLT3-ITD-negative ones (median: 0.9 vs. 0.6 logs/month, Wilcoxon rank sum test, P = 0.041). Peripheral blood and bone marrow were equally useful for minimal residual disease (MRD) detection, and thus, we found that with sampling intervals of 2 months, 94% of relapses would be detected with a median time from MRD detection to hematological relapse of 64 d. In conclusion, this data provide algorithms for handling the rare patients with DEK-NUP214-positive AML allowing for planning of both MRD follow-up and, upon molecular relapse, the timing of cytoreduction or possibly transplant procedures.
Asunto(s)
Algoritmos , Proteínas Cromosómicas no Histona , Leucemia Mieloide Aguda , Modelos Biológicos , Proteínas de Complejo Poro Nuclear , Proteínas de Fusión Oncogénica , Proteínas Oncogénicas , Adolescente , Adulto , Anciano , Niño , Preescolar , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Humanos Par 6/genética , Cromosomas Humanos Par 6/metabolismo , Cromosomas Humanos Par 9/genética , Cromosomas Humanos Par 9/metabolismo , Supervivencia sin Enfermedad , Femenino , Estudios de Seguimiento , Humanos , Cinética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/terapia , Masculino , Persona de Mediana Edad , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa , Recurrencia , Tasa de Supervivencia , Translocación GenéticaRESUMEN
Correct action upon re-emergence of minimal residual disease in acute myeloid leukaemia (AML) patients has not yet been established. The applicability of demethylating agents and use of allogeneic stem cell transplantation will be dependent on pre-relapse AML growth rates. We here delineate molecular growth kinetics of AML harbouring MLL partial tandem duplication (MLL-PTD; 37 cases) compared to those harbouring MLL translocations (43 cases). The kinetics of MLL-PTD relapses was both significantly slower than those of MLL translocation positive ones (median doubling time: MLL-PTD: 24 d, MLL-translocations: 12 d, P = 0·015, Wilcoxon rank sum test), and displayed greater variation depending on additional mutations. Thus, MLL-PTD+ cases with additional RUNX1 mutations or FLT3-internal tandem duplication relapsed significantly faster than cases without one of those two mutations (Wilcoxon rank sum test, P = 0·042). As rapid relapses occurred in all MLL subgroups, frequent sampling are necessary to obtain acceptable relapse detection rates and times from molecular relapse to haematological relapse (blood sampling every second month: MLL-PTD: 75%/50 d; MLL translocations: 85%/25 d). In conclusion, in this cohort relapse kinetics is heavily dependent on AML subtype as well as additional genetic aberrations, with possibly great consequences for the rational choice of pre-emptive therapies.
Asunto(s)
Leucemia Mieloide Aguda/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Secuencias Repetidas en Tándem/genética , Aberraciones Cromosómicas , Análisis Mutacional de ADN/métodos , ADN de Neoplasias/genética , Duplicación de Gen , Genes Relacionados con las Neoplasias/genética , N-Metiltransferasa de Histona-Lisina , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/terapia , Mutación , Neoplasia Residual/genética , Pronóstico , Recurrencia , Translocación GenéticaRESUMEN
The exact disease state of chronic myeloid leukaemia (CML) patients in deep molecular remission is unknown, because even the most sensitive quantitative reverse transcription polymerase chain reaction (qPCR) methods cannot identify patients prone to relapse after treatment withdrawal. To elucidate this, CD34(+) stem cell and progenitor cell subpopulations were isolated by fluorescence-activated cell sorting (FACS), and their content of residual Philadelphia positive (Ph(+) ) cells was evaluated in 17 CML patients (major molecular response, n = 6; 4-log reduction in BCR-ABL1 expression (MR(4) ), n = 11) using both sensitive qPCR and interphase fluorescence in situ hybridization (iFISH). Despite evaluating fewer cells, iFISH proved superior to mRNA-based qPCR in detecting residual Ph(+) stem cells (P = 0·005), and detected Ph(+) stem- and progenitor cells in 9/10 patients at frequencies of 2-14%. Moreover, while all qPCR(+) samples also were iFISH(+) , 9/33 samples were qPCR-/iFISH(+) , including all positive samples from MR(4) patients. Our findings show that residual Ph(+) cells are low BCR-ABL1 producers, and that DNA-based methods are required to assess the content of persisting Ph(+) stem cells in these patients. This approach demonstrates a clinically applicable manner of assessing residual disease at the stem cell level in CML patients in MR(4) , and may enable early and safe identification of candidates for tyrosine kinase inhibitor withdrawal.
Asunto(s)
Proteínas de Fusión bcr-abl/biosíntesis , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Citometría de Flujo/métodos , Proteínas de Fusión bcr-abl/genética , Humanos , Hibridación Fluorescente in Situ/métodos , Interfase , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/terapia , Masculino , Persona de Mediana Edad , Neoplasia Residual , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Real-time quantitative polymerase chain reaction (qPCR) has been extensively validated for the detection of minimal residual disease (MRD) in acute myeloid leukaemia (AML). Meanwhile, multicolour flow cytometry (MFC) has received less attention because the so-called leukaemia-associated immunophenotypes (LAIPs) are generally of lower sensitivity and specificity, and prone to change during therapy. To improve MRD assessment by MFC, we here evaluate the combination of human Myeloid Inhibitory C-type Lectin (hMICL, also termed C-type lectin domain family 12, member A, CLEC12A) and CD 123 (also termed interleukin-3 receptor alpha, IL3RA) in combination with CD34 and CD117 (KIT), as an MRD assay in pre-clinical and clinical testing in 69 AML patients. Spiking experiments revealed that the assay could detect MRD down to 10(-4) in normal bone marrow with sensitivities equalling those of validated qPCR assays. Moreover, it provided at least one MFC MRD marker in 62/69 patients (90%). High levels of hMICL/CD123 LAIPs at the post-induction time-point were a strong prognostic marker for relapse in patients in haematological complete remission (P < 0·001). Finally, in post induction samples, hMICL/CD123 LAIPs were strongly correlated (r = 0·676, P = 0·0008) to applied qPCR targets. We conclude the hMICL/CD123-based MFC assay is a promising MRD tool in AML.
Asunto(s)
Antígenos CD34/metabolismo , Subunidad alfa del Receptor de Interleucina-3/metabolismo , Lectinas Tipo C/metabolismo , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Neoplasia Residual/diagnóstico , Proteínas Proto-Oncogénicas c-kit/metabolismo , Receptores Mitogénicos/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/metabolismo , Médula Ósea/patología , Niño , Femenino , Humanos , Inmunofenotipificación , Leucemia Mieloide Aguda/genética , Masculino , Persona de Mediana Edad , Pronóstico , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Adulto JovenRESUMEN
Chromosome translocation 8q22;21q22 [t(8;21)] is commonly associated with acute myeloid leukemia (AML), and the resulting AML1-ETO fusion proteins are involved in the pathogenesis of AML. To identify novel molecular and therapeutic targets, we performed combined gene expression microarray and promoter occupancy (ChIP-chip) profiling using Lin(-)/Sca1(-)/cKit(+) cells, the major leukemia cell population, from an AML mouse model induced by AML1-ETO9a (AE9a). Approximately 30% of the identified common targets of microarray and ChIP-chip assays overlap with the human t(8;21)-gene expression molecular signature. CD45, a protein tyrosine phosphatase and a negative regulator of cytokine/growth factor receptor and JAK/STAT signaling, is among those targets. Its expression is substantially down-regulated in leukemia cells. Consequently, JAK/STAT signaling is enhanced. Re-expression of CD45 suppresses JAK/STAT activation, delays leukemia development, and promotes apoptosis of t(8;21)-positive cells. This study demonstrates the benefit of combining gene expression and promoter occupancy profiling assays to identify molecular and potential therapeutic targets in human cancers and describes a previously unappreciated signaling pathway involving t(8;21) fusion proteins, CD45, and JAK/STAT, which could be a potential novel target for treating t(8;21) AML.
Asunto(s)
Cromosomas Humanos Par 21/genética , Cromosomas Humanos Par 8/genética , ADN de Neoplasias/metabolismo , Perfilación de la Expresión Génica , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Translocación Genética , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Inmunoprecipitación de Cromatina , Activación Enzimática , Regulación Leucémica de la Expresión Génica , Redes Reguladoras de Genes/genética , Genes Relacionados con las Neoplasias/genética , Humanos , Quinasas Janus/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/genética , Reproducibilidad de los Resultados , Factores de Transcripción STAT/metabolismo , Transducción de Señal/genéticaRESUMEN
Next-generation sequencing (NGS) is an excellent methodology for measuring residual disease in acute myeloid leukemia and surveying several subclones simultaneously. There is little experience with interpretation of differential clonal responses to therapy. We hypothesized that differential clonal response could best be studied in patients with residual disease at the time of response evaluation. We performed targeted panel sequencing of paired diagnostic and first treatment evaluation samples in 69 patients with residual disease by morphology or measurable residual disease (MRD) level >0.02. Five patients had a rising clone at the time of evaluation. In a representative case, the rising clone was present only in the putative healthy stem cells (CD45lowCD34+CD38-CD123-CD7-) and not in the putative leukemic stem cells (CD34+CD38-CD123+CD7+) cells, thus indicating nonmalignant clonal hematopoiesis. In contrast, 17 of 43 evaluable patients exhibited a differential response in genes related to the leukemic clone. Twenty-six of 43 patients exhibited a clonal response that followed the overall treatment response. Patients with a differential response had better event-free survival (EFS) and overall survival (OS) than those in whom the clonal response followed the overall response (log-rank test, EFS: p = 0.045, OS: p = 0.050). This indicates that when following multiple leukemia-related clones, the less chemotherapy-responsive clone could, in some cases, have lower relapse potential, contrary to what is known when using standard mutation or fusion transcript-based disease surveillance. In conclusion, our results confirm the potential of refining MRD assessments by following multiple clones and warrants further studies on the precise interpretations of multiclone NGS-MRD assays.
Asunto(s)
Subunidad alfa del Receptor de Interleucina-3 , Leucemia Mieloide Aguda , Antígenos CD34 , Hematopoyesis Clonal , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Neoplasia ResidualRESUMEN
Overexpressed genes may be useful for monitoring of measurable residual disease (MRD) in patients with childhood acute myeloid leukemia (AML) without a leukemia-specific target. The normal expression of five leukemia-associated genes (SPAG6, ST18, MSLN, PRAME, XAGE1A) was defined in children without hematologic disease (n = 53) and children with suspected infection (n = 90). Gene expression at AML diagnosis (n=50) and during follow-up (n = 21) was compared with child-specific reference values. At diagnosis, 34/50 children (68%) had high expression of at least one of the five genes, and so did 16/31 children (52%) without a leukemia-specific target. Gene expression was quantified in 110 peripheral blood (PB) samples (median, five samples/patient; range, 1 to 10) during follow-up in 21 patients with high expression at diagnosis. All nine patients with PB sampling performed within 100 days of disease recurrence displayed overexpression of SPAG6, ST18, PRAME, or XAGE1A at a median of 2 months (range, 0.6 to 9.6 months) before hematologic relapse, whereas MSLN did not reach expression above normal prior to hematologic relapse. Only 1 of 130 (0.8%) follow-up analyses performed in 10 patients in continuous complete remission had transient expression above normal. SPAG6, ST18, PRAME, and XAGE1A expression in PB may predict relapse in childhood AML patients and facilitate MRD monitoring in most patients without a leukemia-specific target.
Asunto(s)
Antígenos de Neoplasias/genética , Leucemia Mieloide Aguda/genética , Proteínas de Microtúbulos/genética , Proteínas Represoras/genética , Adolescente , Antígenos de Neoplasias/sangre , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Estudios de Casos y Controles , Niño , Preescolar , Femenino , Regulación Leucémica de la Expresión Génica , Humanos , Lactante , Infecciones/sangre , Infecciones/genética , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/terapia , Masculino , Proteínas de Microtúbulos/sangre , Neoplasia Residual , Proteínas Represoras/sangreRESUMEN
In acute myeloid leukemia (AML) mouse models, the RUNX1-RUNX1T1 fusion protein has failed to produce leukemia by itself, but alternative splicing of exon 9a of the RUNX1-RUNX1T1 fusion transcript (FT) has recently been shown to enhance the leukemogenic potential. We have analyzed 138 diagnosis and follow-up samples from 13 RUNX1-RUNX1T1+ patients as well as diagnosis samples from 13 RUNX1-RUNX1T1- AML patients and 26 healthy donors. Levels of native RUNX1T1 mRNA were low in both healthy and RUNX1-RUNX1T1-negative AML samples. Likewise, the ratio between RUNX1T1 mRNA harboring exon 9a and lacking exon 9a was low and tightly regulated (0.017-0.11). In contrast, 11/13 RUNX1-RUNX1T1-positive AML patients displayed high and variable ratios of FT ranging from 0.05 to 0.46 (P < 0.001, Wilcoxon rank-sum test), indicating altered exon 9a splicing in these patients. Importantly, patients who remained in continuous complete remission displayed a faster disappearance of the RUNX1-RUNX1T1 exon 9a splice variant compared to patients bound to relapse (P = 0.02). In conclusion, alternative splicing seems to be part of the leukemogenic process in the majority of RUNX1-RUNX1T1-positive AML patients, and splice variant kinetics under cytoreduction may be a predictor for patients prone to relapse.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/biosíntesis , Exones , Leucemia Mieloide Aguda/metabolismo , Proteínas de Fusión Oncogénica/biosíntesis , Empalme del ARN , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesis , Adolescente , Adulto , Animales , Secuencia de Bases , Niño , Preescolar , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Femenino , Estudios de Seguimiento , Humanos , Leucemia Mieloide Aguda/genética , Masculino , Ratones , Persona de Mediana Edad , Proteínas de Fusión Oncogénica/genética , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/genética , ARN Neoplásico/genética , Proteína 1 Compañera de Translocación de RUNX1 , Recurrencia , Inducción de Remisión , Eliminación de Secuencia , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
The criteria to evaluate response to treatment in acute myeloid leukemia (AML) have changed little in the past 60 years. It is now possible to use higher sensitivity tools to measure residual disease burden in AML. Such minimal or measurable residual disease (MRD) measurements provide a deeper understanding of current patient status and allow stratification for risk of subsequent clinical relapse. Despite these obvious advantages, and after over a decade of laboratory investigation and preclinical validation, MRD measurements are not currently routinely used for clinical decision-making or drug development in non-acute promyelocytic leukemia (non-APL) AML. We review here some potential constraints that may have delayed adoption, including a natural hesitancy of end users, economic impact concerns, misperceptions regarding the meaning of and need for assay sensitivity, the lack of one single MRD solution for all AML patients, and finally the need to involve patients in decision-making based on such correlates. It is our opinion that none of these issues represent insurmountable barriers and our hope is that by providing potential solutions we can help map a path forward to a future where our patients will be offered personalized treatment plans based on the amount of AML they have left remaining to treat.