Fluorescence in situ hybridization: a highly efficient technique of molecular diagnosis and predication for disease course in patients with myeloid leukemias

Abstract

The accuracy of cytogenetic diagnosis in the management of hematological malignancies has improved significantly over the past 10 years. Fluorescence in situ hybridization (FISH), a technique of molecular cytogenetics, has played a pivotal role in the detection of unique sub-microscopic chromosomal rearrangements that helped in the identification of chromosomal loci, which contain genes involved in leukemogenesis. We studied the feasibility and sensitivity of the FISH technique for molecular analysis of translocations markers, t(9;22) and t(15;17) for accurate molecular diagnosis and for monitoring the disease in 21 patients with chronic myeloid leukemia (CML) who received interferon-α and/or chemotherapy (7 patients), bone marrow transplantation (14 patients), and 14 patients with acute promyelocytic leukemia (APL) who received all-trans-retinoic acid (ATRA) and/or chemotherapy. We also applied conventional karyotyping (CK) for identification of t(9;22) and t(15;17) at diagnosis. All CML cases had a Ph; t(9;22) and except for two cases all APL had t(15;17). The FISH studies on CML marrows in complete cytogenetic remission (CCR) (100% Ph− by CK) achieved by IFN-α, showed 0–2.5% of cells with BCR-ABL fusion in first cytogenetic remission (Controls, range 0.5–1.5%). Repeat follow-up FISH studies could be done in two cases in remission, which demonstrated 0–10% of cells with BCR-ABL fusion. Evaluation of Ph positive status of CML marrow at diagnosis by CK (100% Ph+ cells) and FISH (80–92% BCR-ABL fusion) pointed the existence of dormant clone of normal residual hematopoietic cells along with actively proliferating clones of Ph positive cells. Flourescence in situ hybridization analysis of post-BMT CML marrows in CCR (0% Ph+ mitoses) could detect MRD with range of 1–6%. Among 14 patients, 9 who showed percentage of BCR-ABL positive cells (0.0–1.5%) almost similar to normal controls, 6 patients had comparatively good prognosis (disease-free survival 7–14 months). Of five patients with residual leukemic cells in the range of 2–6%, 4 relapsed within a period of 3–24 months. Fourteen APL patients in CCR [100% t(15;17) negative cells by CK] were evaluated by FISH to check the presence of residual leukemic cells. In these patients FISH could efficiently detect 1–14.5% of residual cells with PML-RARA (patients mean MRD 5%, controls mean MRD 3.5%, P=.02). Since the time of FISH analysis, 5 to 7 patients with higher fraction of leukemic cells (5–11%) relapsed within a short period (1–7 months). On the contrary, 5 of 7 patients with either absence or low percentage of PML-RARA positive cells remained in complete remission for 11–24 months. Our data show that FISH has a potential to detect and measure the fraction of aberrant malignant cells in remission marrows, induced by BMT in CML and chemotherapy in APL. These findings encourage the investigations on a large scale to merit its potential for identification of patients at high risk. In the present studies, FISH on interphase cells also demonstrated its efficiency in the molecular diagnosis by its ability to detect BCR-ABL and PML-RARA fusion in CML with masked/variant Ph and t(15;17) negative APL, respectively. The efficiency of technique in molecular diagnosis was also proved in one of the CML patients who progressed to myeloid blastic phase where interphase FISH could identify an extra BCR-ABL fusion on both chromosomes 9 indicating insertion of BCR into ABL and its duplication.