טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentZaatreh Maria
SubjectIdentification of Genetic Alterations and its Relation to
Differentiation Markers that Distinguish
Subpopulations of leukemic Cells in AML
Patients
DepartmentDepartment of Medicine
Supervisor Dr. Yishai Ofran


Abstract

Acute myeloid leukemia consists of heterogeneous mixture of genetically distinct sub-clones with different leukemogenic potential and thus different active pathways may affect cells’ response to therapy. These leukemic sub-populations may also vary in their sensitivity to chemotherapy. It is well known that myeloid leukemia cells express variable and different immunophenotypes that dynamically being changed along therapy. However, how changes in immunophenotype correlate with the genetic heterogeneity is not clearly understood. Recently it was recognized that as early as the first induction treatment, inherent chemo-resistant sub-populations are already exist, survive the chemotherapy, and may become dominant at relapse.

In order to explore association of sub-clonal heterogeneity with differentiation capacity and with chemotherapy sensitivity, we separated patients’ leukemia cells based on CD34 expression into differentiated and undifferentiated populations. Genes known to be mutated in AML were sequenced using targeted deep sequencing in order to monitor changes in mutation allele frequencies of each distinguished cell population. Mutation allele frequency reflects the impartial part of the clone carrying the mutation from the studied cells which were harvested according to their differentiation stage. Revealing mutational allele frequencies in different populations of 4 acute myeloid leukemia patients demonstrated different distribution of sub-clones. Certain sub-clones tend to be more prevalent than others in undifferentiated populations and therefore assumed to be more resistant to chemotherapy. However, no direct relation between sub-clones and differentiation markers was learned.

A myeloid leukemia cell line (Kasumi) was separated into two cell populations -as the patients' samples by its CD34 expression - in order to examine cell viability following chemotherapy exposure. Sorted cells exposed in vitro to different doses of chemotherapy, demonstrated that undifferentiated cell population survives chemotherapy and is more resistant than differentiated population.

In conclusion, not only leukemic stem cell but also specific sub-clones may be less likely to differentiate and therefore relatively resistant to chemotherapy, even without the presence of specific mutation in genes directly involved in chemotherapy induced cell death mechanism.