ABSTRACT
Juvenile myelomonocytic leukemia (JMML) is a rare myeloproliferative disease of early childhood that develops due to mutations in the genes of the RAS-signaling pathway. Next-generation high throughput sequencing (NGS) enables identification of various secondary molecular genetic events that can facilitate JMML progression and transformation into secondary acute myeloid leukemia (sAML). The methods of single-cell DNA sequencing (scDNA-seq) enable overcoming limitations of bulk NGS and exploring genetic heterogeneity at the level of individual cells, which can help in a better understanding of the mechanisms leading to JMML progression and provide an opportunity to evaluate the response of leukemia to therapy. In the present work, we applied a two-step droplet microfluidics approach to detect DNA alterations among thousands of single cells and to analyze clonal dynamics in two JMML patients with sAML transformation before and after hematopoietic stem cell transplantation (HSCT). At the time of diagnosis both of our patients harbored only "canonical" mutations in the RAS signaling pathway genes detected by targeted DNA sequencing. Analysis of samples from the time of transformation JMML to sAML revealed additional genetic events that are potential drivers for disease progression in both patients. ScDNA-seq was able to measure of chimerism level and detect a residual tumor clone in the second patient after HSCT (sensitivity of less than 0.1% tumor cells). The data obtained demonstrate the value of scDNA-seq to assess the clonal evolution of JMML to sAML, response to therapy and engraftment monitoring.
Subject(s)
Leukemia, Myelomonocytic, Juvenile , Humans , Child, Preschool , Leukemia, Myelomonocytic, Juvenile/genetics , Leukemia, Myelomonocytic, Juvenile/therapy , Clonal Evolution , High-Throughput Nucleotide Sequencing , Mutation/geneticsABSTRACT
IPSC line RCPCMi004-A was generated from skin fibroblasts collected from a male patient with early onset Parkinson's disease. The patient carries a heterozygous deletion of the exon 2 of PARK2 gene. The reprogramming of fibroblasts was performed with Sendai viruses containing Oct-4, Sox-2, Klf-4 and c-Myc. Pluripotency was confirmed by immunofluorescence, RT-PCR, and formation of embryoid bodies. The RCPCMi004-A cell line carries the same deletion in PARK2 gene. The RCPCMi004-A cell line can be used to model Parkinson's disease in vitro.
Subject(s)
Induced Pluripotent Stem Cells , Parkinson Disease , Cell Differentiation , Cell Line , Embryoid Bodies , Exons/genetics , Humans , Male , Parkinson Disease/geneticsABSTRACT
Classical osteosarcoma is one of the most common primary malignant bone tumors in children and adolescents. It more frequently occurs in the areas of the highest growth plate proliferation: limb long bones particularly in the distal femur (30%), proximal tibia (15%), and proximal humerus (15%). In the long bones, the tumor is located usually in the metaphysis (90%), less frequently in the diaphysis (9%), and very rarely in the epiphysis. This paper considers the clinical, radiological, and histological diagnosis, and prognosis in this pathology.