Diagnosis of chronic myeloid and acute lymphocytic leukemias by detection of leukemia-specific mRNA sequences amplified in vitro.

Abstract

The Philadelphia chromosome is present in more than 95% of chronic myeloid leukemia patients and 13% of acute lymphocytic leukemia patients. The Philadelphia translocation, t(9;22), fuses the BCR and ABL genes resulting in the expression of leukemia-specific, chimeric BCR-ABL messenger RNAs. To facilitate diagnosis of these leukemias, we have developed a method of amplifying and detecting only the unique mRNA sequences, using an extension of the polymerase chain reaction technique. Diagnosis of chronic myeloid and acute lymphocytic leukemias by this procedure is rapid, much more sensitive than existing protocols, and independent of the presence or absence of an identifiable Philadelphia chromosome.

Overexpression of tumor necrosis factor (TNF)-α and interferon (IFN)-γ by bone marrow cells from patients with myelodysplastic syndromes

“…The PCR reaction was performed as described elsewhere. 21,23,24 As a control reaction of PCR, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (196 bp) was also detected in each run. Briefly, 50 ng of the RNA was used for RT-PCR.…”

“…23,24 This study therefore attempted to investigate the disturbed regulatory mechanisms of hematopoiesis in MDS in vivo by defining up-regulation of inhibitory cytokine (TNF-␣ and IFN-␥) production using RT-PCR and then to immunohistochemically demonstrate the localization of the cytokine producing cells in the bone marrow.…”

A novel BCR-ABL rearrangement in a Philadelphia chromosome-positive chronic myelogenous leukaemia variant with thrombocythaemia

“…Inner primers and amplification conditions were as described. 7 The number of cycles were 31 and 36, respectively. The RT-PCR-amplified fragments were electrophoresed and visualised with ethidium bromide on a 2% agarose gel.…”

“…Southern blot hybridisation was performed using digoxigenin-labelled probes (Boehringer Mannheim, Penzberg, Germany) complementary to either the b2a2 or b3a2 junctions of the BCR/ABL fusion gene. 7 The amplicons were sequenced using an ABI 373 sequencer (Applied Biosystems, Foster City, CA, USA).…”

“…6 The commonest BCR/ABL fusion proteins result from a b2a2 or b3a2 gene fusion and this simplifies the detection of the Philadelphia translocation by genetic technology using a conventional set of gene fusion probes. 7 A patient is reported who presented clinically as essential thrombocythaemia. On the basis of the karyotypic finding of a Philadelphia chromosome she was diagnosed and treated as a CML variant, but with weak hybridization signals using conventional probes, and in whom molecular genetic analysis showed an unusually constituted BCR-ABL rearrangement.…”