A comparative analysis of FISH, RT-PCR, and cytogenetics for the diagnosis of bcr-abl-positive leukemias.

Philadelphia (Ph) chromosome-positive leukemias, with the bcr-abl gene translocation, have a dismal prognosis. The identification of Ph-positive patients is vitally important because only aggressive therapeutic approaches, such as allogeneic bone marrow transplantation, may result in long-term disease-free survival. Routine diagnostic methods, such as Southern blot analysis and cytogenetics, may lead to false-negative results. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis is considered the most sensitive tool for the detection of the bcr-abl translocation, and it is widely used alone or in combination with karyotyping or Southern blot analysis to identify Ph-positive cases. In this study, we used fluorescence in situ hybridization (FISH) with BCR and ABL double-color probes for detecting Ph-positive leukemias. The FISH results were compared with the results of cytogenetic and RT-PCR analyses in 75 patients with leukemia or other myeloproliferative syndromes (chronic myeloid leukemia, 30; acute lymphoblastic leukemia, 24; acute myelogenous leukemia, 6; essential (hemorrhagic) thrombocythemia, 12; chronic myelomonocytic leukemia, 2; and polycythemia vera, 1). FISH analysis proved to be simple, extremely reliable and sensitive; bcr-abl fusion detection was successful in the presence of all types of molecular junctions i.e., (b2a2, b3a2, and e1a2). Furthermore, a Ph-positive case that proved fusion negative by RT-PCR was identified as positive by FISH. The sensitivity of RT-PCR and FISH related to Ph-positive cases were 97% and 100%, respectively. Regarding specificity, in 4 (5%) of 75 patients, RT-PCR provided false-positive results. Cross-contamination was identified because a new specimen was harvested and reanalyzed when FISH, cytogenetics, and RT-PCR results were contradictory. We believe FISH is an optimal diagnostic method to detect bcr-abl translocation that can be used alone or to validate the results of RT-PCR analysis.

Effect of bcr-abl oligodeoxynucleotides on the clonogenic growth of chronic myelogenous leukaemia cells.

We studied the effect of phosphorothioate oligodeoxynucleotides ([S]ODNs) complementary to the bcr-abl junction on cells taken at diagnosis from 41 patients with Philadelphia-positive chronic myelogenous leukaemia (CML). Experiments included the evaluation of the anti-leukaemic effect of 16- and 26-mer antisense [S]ODNs on both mononuclear and CD34+ cells, evaluation of incubation time and correlation of colony growth inhibition with the down-regulation of p210(bcr-abl). At the same time, the uptake of [S]ODNs by mononuclear and purified CD34+ cell populations and the cross-hybridization of 26- and 16-mer [S]ODNs with the complementary sequences were evaluated. After incubation for 120 h with 26-mer antisense [S]ODNs on mononuclear cells, overall mean colony recovery was 41.9% of the untreated control samples; in particular, a significant reduction in colony formation was observed in 22 of the 35 cases tested. The effect of 26-mer ODNs on CD34+ cells was comparable to that observed on mononuclear cells in terms of colony inhibition; however, a higher proportion of cases showed a significant inhibition of colony formation. In comparison with the 26-mer antisense [S]ODNs, the anti-leukaemic effect of the 16-mer antisense [S]ODNs was less evident on mononuclear cells and comparable on CD34+ cells; however, a more specific effect was evident on both target cells. Hybridization experiments confirmed a partial cross-reactivity when the 26-mer ODNs were hybridized with their complementary sequence; this did not occur when 16-mer ODNs were similarly tested. Experiments aimed at evaluating the effect of the incubation time showed a significant increase in anti-leukaemic effect after a 120 h incubation period compared to that measured after a 24 h incubation period; this was parallelled by a progressive increase in the intracellular concentrations of [S]ODNs from day 1 to day 5. The accumulation of [S]ODNs correlated with a marked down-regulation of p210(bcr-abl) levels which was first detectable after 72 h of treatment. The down-regulation of p210(bcr-abl) levels following treatment with [S]ODNs showed a correlation between the effect of antisense [S]ODNs on leukaemic colony formation and protein expression. These studies confirm that, under optimal conditions of target cell culture and ODN size, antisense [S]ODNs complementary to the bcr-abl junction have specific anti-leukaemic effects.

Sequential molecular monitoring of chimerism in chronic myeloid leukemia patients receiving donor lymphocyte transfusion for relapse after bone marrow transplantation.

Recent observations of chimerism in patients relapsed following an allotransplant suggest the persistence of immunotolerance, thus offering a biologic rationale for the use of donor lymphocyte transfusion (DLT). In this study, we have analyzed by PCR amplification of several VNTR regions, sequential bone marrow and peripheral blood DNA samples in four patients who received DLT for CML relapse after bone marrow transplantation. Prior to DLT, all patients showed mixed chimerism in peripheral blood cells while two had mixed chimerism and two no chimerism in the BM. None of these four patients showed evidence of chimerism at the cytogenetic level (all had 100% +ve metaphases). After DLT, a complete hematologic and molecular remission (ie disappearance of the BCR/ABL fusion transcript) was obtained in the two patients who had bone marrow mixed chimerism prior to DLT. The two patients without evidence of marrow chimerism prior to DLT converted to a pattern of mixed chimerism after DLT, but both developed a severe bone marrow aplasia occurring at day 56 and 36, respectively. With regard to the sequential analysis of bone marrow chimerism after DLT we observed that: (1) the disappearance of BCR/ABL +ve cells paralleled the conversion to a pattern of full donor chimerism; and (2) the time interval to achieve CR was inversely correlated with the percentage of donor DNA in bone marrow. In conclusion, we have shown here that the assessment of bone marrow pre-DLT chimerism by PCR analysis might predict the response in patients with favorable characteristics, and also might identify patients at high risk of developing severe myelosuppression.

Improved rapid detection of the PML/RARalpha fusion gene in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is a medical emergency which requires rapid diagnosis and tailored treatment. Detection of the PML/RARalpha fusion gene in APL blasts is critical to start promptly the specific therapy with all-trans retinoic acid (ATRA). APL lacking this genetic lesion have been reported as being ATRA resistant. Reverse-transcription polymerase chain reaction (RT-PCR) has been extensively used to detect the PML/RARalpha cDNA. The reported PML/RARalpha amplification techniques are laborious and time consuming, and include conventional RNA extraction, cDNA synthesis and a two-round (nested) PCR. We hereby describe a few variations of the commonly adopted RNA extraction and PML/RARalpha RT-PCR protocols which allow a molecular diagnosis of APL to be carried out in less than 5 h. Processing of small volumes of leukemic cell lysate (0.5 ml) in a microfuge allows extraction of good quality RNA in 1 h. After reverse transcription to obtain cDNA, a 'hot start' PCR procedure was adopted which enabled us to amplify clearly visible and specific products after a single (not nested) amplification round. The PML/RARalpha fusion gene was detected in the blasts of six consecutive APL at diagnosis, and an APL-tailored protocol including ATRA was started in each case within 6 h of admission. On repeated experiments, the assay proved highly specific and sensitive for the rapid detection of all PML/RARalpha transcript types. Our data should encourage the use of this rapid procedure for the diagnosis of both typical APL and, particularly, less typical cases awaiting urgent therapeutic intervention.