Integrated flow cytometry and sequencing to reconstruct evolutionary patterns from dysplasia to acute myeloid leukemia

Clonal evolution in acute myeloid leukemia (AML) originates long before diagnosis and is a dynamic process that may affect survival. However, it remains uninvestigated during routine diagnostic workup. We hypothesized that the mutational status of bone marrow dysplastic cells and leukemic blasts, analyzed at the onset of AML using integrated multidimensional flow cytometry (MFC) immunophenotyping and sorting (FACS) with next-generation sequencing (NGS), could reconstruct leukemogenesis. Dysplastic cells were detected by MFC in 285 of 348 (82%) newly-diagnosed AML patients. Presence of dysplasia according to MFC and WHO criteria had no prognostic value in the elderly. NGS of dysplastic cells and blasts isolated at diagnosis identified three evolutionary patterns: stable (n=12/21), branching (n=4/21) and clonal evolution (n=5/21). In patients achieving complete response, integrated MFC and FACS with NGS showed persistent measurable residual disease (MRD) in phenotypically normal cell types, as well as the acquisition of genetic traits associated with treatment resistance. Furthermore, whole-exome sequencing of dysplastic and leukemic cells at diagnosis and of MRD uncovered different clonal involvement in dysplastic myelo-erythropoiesis, leukemic transformation and chemoresistance. Altogether, we showed that it is possible to reconstruct leukemogenesis in approximately 80% of newly diagnosed AML patients, using techniques other than single-cell multiomics.ACKNOWLEDGEMENTS: The authors acknowledge the patients, caregivers, and the biobank of the University of Navarra. This work was supported by grants from the Área de Oncología del Instituto de Salud Carlos III, Centro de Investigacion Biom ´ edica en ´ Red (CIBER-ONC) (CB16/12/00369, CB16/12/00233, CB16/12/ 00489, and CB16/12/00284), Instituto de Salud Carlos III/Subdireccion General de Investigaci ´ on Sanitaria (FIS numbers PI16/ ´ 01661, PI16/00517, and PI19/01518), and the Plan de Investigacion´ de la Universidad de Navarra (PIUNA 2014-18). This work was supported internationally by the Cancer Research UK, FCAECC, and AIRC under the Accelerator Award Program (EDITOR)

In vitro culture with interleukin-15 leads to expression of activating receptors and recovery of natural killer cell function in acute myeloid leukemia patients

A pesar de los avances recientes en el abordaje terapéutico de las hemopatías malignas, sus pronósticos siguen siendo con frecuencia deficientes. La inmunoterapia podría abrir una nueva ventana de gran interés en este contexto. Las células asesinas naturales (NK) constituyen un área importante de investigación para las neoplasias malignas hematológicas, ya que esta subpoblación puede matar células diana de forma espontánea sin sensibilización previa, lo que representa una herramienta novedosa en el tratamiento de ellas. Se observa una función citolítica NK anormal en varias neoplasias malignas hematológicas, incluida la leucemia mieloide aguda (LMA) y los síndromes mielodisplásicos. Varios mecanismos están involucrados en esta función anormal, como la disminución de la expresión de los receptores de activación, el aumento de la expresión de los receptores inhibidores o la expresión defectuosa de los ligandos de las células NK en las células diana. Las nuevas inmunoterapias se centran en identificar factores que podrían aumentar la expresión de estos receptores activadores, para contrarrestar la expresión de los receptores inhibidores y, por lo tanto, mejorar las capacidades citotóxicas de las células NK contra las células tumorales. En este trabajo, analizamos el efecto de la interleucina (IL) -15 en la expresión de los receptores activadores de células NK que desempeñan un papel crucial en la lisis de blastos de pacientes con AML. Nuestros resultados mostraron que la IL-15 aumentó la expresión superficial de NKp30 en células NK de donantes sanos y pacientes con AML, con la consiguiente mejora de la citotoxicidad de las células NK. Además, la regulación positiva de NKp30 inducida por la IL-15 se asocia con una mejora de la maduración de las células dendríticas (DC) mieloides mediadas por NK. Las células NK cultivadas con IL-15 mostraron una regulación positiva de NKp30, que se asocia con un aumento de la actividad antitumoral y con una maduración mejorada de las DC inmaduras. En nuestro modelo in vitro, la IL-15 ejerció un gran estímulo activador que podría usarse como nueva inmunoterapia en pacientes con AML.Despite recent progress in the therapeutic approach of malignant hemopathies, their prognoses remain frequently poor. Immunotherapy could open a new window of great interest in this setting. Natural killer (NK) cells constitute an important area of research for hematologic malignancies, because this subpopulation is able to kill target cells spontaneously without previous sensitization, representing a novel tool in the treatment of them. Abnormal NK cytolytic function is observed in several hematological malignancies, including acute myeloid leukemia (AML) and myelodysplastic syndromes. Several mechanisms are involved in this abnormal function, such as decreased expression of activating receptors, increased expression of inhibitory receptors or defective expression of NK cell ligands on target cells. New immunotherapies are focused in identifying factors that could increase the expression of these activating receptors, to counteract inhibitory receptors expression, and therefore, to improve the NK cell cytotoxic capacities against tumor cells. In this work, we analyze the effect of interleukin (IL)-15 on the expression of NK cell-activating receptors that play a crucial role in the lysis of blasts from AML patients. Our results showed that IL-15 increased the surface expression of NKp30 on NK cells from healthy donors and AML patients with the consequent improvement of NK cell cytotoxicity. Besides, the upregulation of NKp30 induced by IL-15 is associated with an improvement of NK-mediated myeloid dendritic cells (DCs) maturation. NK cells cultured with IL-15 showed an upregulation of NKp30, which is associated with an increase anti-tumor activity and with an improved maturation of immature DCs. In our in vitro model, IL-15 exerted a great activating stimulus that could be used as novel immunotherapy in AML patients.• Ministerio de Economía y Competitividad. Beca SAF2013-46161-R, para Raquel Tarazona Lafarga • Junta de Extremadura. Beca IB16164, para Raquel Tarazona Lafarga • Ministerio de Salud. Beca PI13/02691 y PI16/01615, para Rafael Solana Lara • Junta de Andalucía. Ayuda CTS-208 • Junta de Extremadura y Fondos Europeos de Desarrollo Regional. Beca GR15183, para Raquel Tarazona Lafarga • Universidad de Extremadura y Fondos Europeos de Desarrollo Regional. Ayuda para Raquel Tarazona LafargapeerReviewe