To understand the cytogenetic mechanisms responsible for multiple RUNX1 gene copy numbers in hematologic malignancies, we analyzed the chromosomal and molecular cytogenetic findings in bone marrow or peripheral blood samples of individuals who were diagnosed with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), or acute lymphoblastic leukemia (ALL). Included in the analysis were 113 consecutive samples received in our laboratory between January 2005 and June 2007. Bone marrow and/or peripheral blood samples were characterized using conventional G-banding techniques and fluorescent in situ hybridization (FISH) techniques with commercially available RUNX1/RUNX1T1 or ETV6/RUNX1 dual-color fusion probes. Eighty-one (72%) of the 113 samples showed an abnormal karyotype and/or abnormal FISH results. Eight of these had, by interphase FISH, RUNX1/RUNX1T1 or RUNX1/ETV6 fusion, and 19 had three or more RUNX1 signals not related to fusion with RUNX1T1 or ETV6 gene. Of the 19 cases with multiple RUNX1 gene signals, 5 had high-level RUNX1 amplification--defined as 5 or more RUNX1 signals in interphase cells--whereas the remaining 14 had 3-4 RUNX1 signals. Four of the five tumors with high-level RUNX1 amplification were myeloid disorders--three cases of AML and one case of MDS. The karyotypes of tumors with high-level amplification of RUNX1 were significantly characterized by the presence of marker chromosomes that harbored extra copies of the RUNX1 gene compared with tumors that had three to four RUNX1 gene signals (P=0.026, Fisher's exact test). Our findings show that high-level RUNX1 amplification, especially in myeloid disorders, often results from marker chromosomes harboring extra copies of the RUNX1 gene . This suggests that amplification of RUNX1 in these tumors may be secondary to a previous rearrangement of 21q22, which later evolved into a complex marker chromosome as part of tumor progression.