A novel method for single cell detection of in situ telomerase or histone H3 in combination with clonal analysis by FISH

Abstract 

A novel method for simultaneously detecting clonality by FISH, and presence of telomerase activity (telo+ cells) or histone H3 mRNA (H3+) in single cells from a mixed leukemic population is reported. The methods were validated using K562 cells mixed with peripheral blood granulocytes and bone marrow aspirate cells from newly diagnosed AML patients. Fifty patients with AML were analyzed for telo+ cells, while eight AML patients were analyzed for FISH-Telomerase and FISH-H3+ during remission induction therapy. Our results demonstrate that: (1) changes in the leukemic populations during therapy could be followed; (2) a favorable response to chemotherapy occurred when there was a reduction in both the cytogenetically abnormal cells along with reduction in telo+ cells within this abnormal population; (3) reduction of either telo+ cells or FISH+ cells alone did not correlate with good response. H3+ could be detected in only 4% of the leukemic population, most of which were cytogenetically abnormal. These newly established methods allow sub-populations of cells to be followed during disease progression and treatment and to elucidate factors that give a specific clone proliferative advantage.

Keywords: Telomerase, Histone H3, FISH, In situ PCR, Leukemia