Assuntos
Tirosina Quinase da Agamaglobulinemia/genética , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Fosfolipase C gama/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Adenina/análogos & derivados , Adenina/uso terapêutico , Idoso , Antineoplásicos/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Piperidinas/uso terapêutico , Sulfonamidas/uso terapêuticoRESUMO
The existence of "leukemia-initiating cells" (LICs) in chronic lymphocytic leukemia (CLL) remains controversial due to the difficulty in isolating and identifying the tumor-initiating cells. Here, we demonstrate a microchannel electroporation (MEP) microarray that injects RNA-detecting probes into single live cells, allowing the imaging and characterization of heterogeneous LICs by intracellular RNA expression. Using limited-cell FACS sequencing (LC-FACSeq), we can detect and monitor rare live LICs during leukemogenesis and characterize their differential drug sensitivity. Disease-associated mutation accumulation in developing B lymphoid but not myeloid lineage in CLL patient hematopoietic stem cells (CLL-HSCs), and development of independent clonal CLL-like cells in murine patient-derived xenograft models, suggests the existence of CLL LICs. Furthermore, we identify differential protein ubiquitination and unfolding response signatures in GATA2high CLL-HSCs that exhibit increased sensitivity to lenalidomide and resistance to fludarabine compared to GATA2lowCLL-HSCs. These results highlight the existence of therapeutically targetable disease precursors in CLL.
Assuntos
Leucemia Linfocítica Crônica de Células B , Animais , Células Cultivadas , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Camundongos , Células-Tronco Neoplásicas/metabolismo , RNA/metabolismoRESUMO
Hematopoiesis is hierarchical, and it has been postulated that acute myeloid leukemia (AML) is organized similarly with leukemia stem cells (LSCs) residing at the apex. Limited cells acquired by fluorescence activated cell sorting in tandem with targeted amplicon-based sequencing (LC-FACSeq) enables identification of mutations in small subpopulations of cells, such as LSCs. Leveraging this, we studied clonal compositions of immunophenotypically-defined compartments in AML through genomic and functional analyses at diagnosis, remission and relapse in 88 AML patients. Mutations involving DNA methylation pathways, transcription factors and spliceosomal machinery did not differ across compartments, while signaling pathway mutations were less frequent in putative LSCs. We also provide insights into TP53-mutated AML by demonstrating stepwise acquisition of mutations beginning from the preleukemic hematopoietic stem cell stage. In 10 analyzed cases, acquisition of additional mutations and del(17p) led to genetic and functional heterogeneity within the LSC pool with subclones harboring varying degrees of clonogenic potential. Finally, we use LC-FACSeq to track clonal evolution in serial samples, which can also be a powerful tool to direct targeted therapy against measurable residual disease. Therefore, studying clinically significant small subpopulations of cells can improve our understanding of AML biology and offers advantages over bulk sequencing to monitor the evolution of disease.
Assuntos
Biomarcadores Tumorais/genética , Evolução Clonal , Genômica/métodos , Células-Tronco Hematopoéticas/patologia , Leucemia Mieloide Aguda/patologia , Mutação , Células-Tronco Neoplásicas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Citometria de Fluxo , Seguimentos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imunofenotipagem , Leucemia Mieloide Aguda/genética , Masculino , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/metabolismo , Prognóstico , Adulto JovemRESUMO
Detecting, characterizing, and monitoring rare populations of cells can increase testing sensitivity, give insight into disease mechanism, and inform clinical decision making. One area that can benefit from increased resolution is management of cancers in clinical remission but with measurable residual disease (MRD) by multicolor FACS. Detecting and monitoring genomic clonal resistance to treatment in the setting of MRD is technically difficult and resource intensive due to the limited amounts of disease cells. Here, we describe limited-cell FACS sequencing (LC-FACSeq), a reproducible, highly sensitive method of characterizing clonal evolution in rare cells relevant to different types of acute and chronic leukemias. We demonstrate the utility of LC-FACSeq for broad multigene gene panels and its application for monitoring sequential acquisition of mutations conferring therapy resistance and clonal evolution in long-term ibrutinib treatment of patients with chronic lymphocytic leukemia. This technique is generalizable for monitoring of other blood and marrow infiltrating cancers.
Assuntos
Adenina/análogos & derivados , Evolução Clonal/imunologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia/tratamento farmacológico , Neoplasia Residual/tratamento farmacológico , Piperidinas/uso terapêutico , Adenina/uso terapêutico , Células Clonais , Humanos , Leucemia/imunologia , Mutação/genética , Neoplasia Residual/diagnósticoAssuntos
Hematopoiese Clonal , Animais de Estimação , Animais , Cães , Hematopoese/genética , Humanos , MutaçãoRESUMO
Purpose Therapeutic targeting of Bruton tyrosine kinase (BTK) with ibrutinib in chronic lymphocytic leukemia has led to a paradigm shift in therapy, and relapse has been uncommon with current follow-up. Acquired mutations in BTK and PLCG2 can cause relapse, but data regarding the prevalence and natural history of these mutations are limited. Patients and Methods Patients accrued to four sequential studies of ibrutinib were included in these analyses. Deep sequencing for BTK and PLCG2 was performed retrospectively on patients who experienced relapse and prospectively on a screening population. Results With a median follow-up time of 3.4 years, the estimated cumulative incidence of progression at 4 years is 19% (95% CI, 14% to 24%). Baseline karyotypic complexity, presence of del(17)(p13.1), and age less than 65 years were risk factors for progression. Among patients who experienced relapse, acquired mutations of BTK or PLCG2 were found in 85% (95% CI, 71% to 94%), and these mutations were detected an estimated median of 9.3 months (95% CI, 7.6 to 11.7 months) before relapse. Of a group of 112 patients examined prospectively, eight patients have experienced relapse, and all of these patients had acquired resistance mutations before relapse. A resistance mutation was detected in an additional eight patients who have not yet met criteria for clinical relapse. Conclusion Relapse of chronic lymphocytic leukemia after ibrutinib is an issue of increasing clinical significance. We show that mutations in BTK and PLCG2 appear early and have the potential to be used as a biomarker for future relapse, suggesting an opportunity for intervention.