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A new complex rearrangement involving chromosome bands 5q13, 12p13, 22q11, and 3q12 was identified and characterized in a patient with acute myeloid leukemia. Fluorescence in situ hybridization showed the involvement of the ETV6 gene in 12p13. ETV6 primers were specifically designed for 3'- and 5'-RACE-PCR experiments, which led to the identification of the other two rearranged genes. The derivative chromosome 5 harbored a fusion of the ETV6 sequence with that of the LOC115548 gene. The two genes were placed in opposite orientation and did not encode a fusion protein. On the derivative chromosome 12, ETV6 was fused to the MN1 gene on chromosome 22. Also in this case, the insertion, within the MN1 sequence, of a portion of chromosome 3 prevented the formation of a fusion protein. Finally, the derivative chromosome 22 contained the 3' portions of both LOC115548 and MN1, and no fusion transcript with coding potential could be predicted. In conclusion, all chromosome breakpoints led to the truncation of the three involved genes in the absence of predicted fusion proteins. This study lends further support to the hypothesis that gene disruption resulting in either loss of function or haploinsufficiency may be relevant in acute myeloid leukemia pathogenesis., (Copyright 2004 Wiley-Liss, Inc.)

We have investigated the expression of the M-CSF receptor (c-fms) in 16 freshly isolated acute promyelocytic leukemias (APL) expressing the PML/RAR alpha fusion protein. In parallel, we evaluated the capacity of these cells to differentiate along the granulocytic and monocytic pathways. c-fms was constitutively and constantly expressed in all cases sensitive in vivo to all-trans retinoic acid (ATRA) and its expression was further potentiated following in vitro induction with ATRA. Furthermore, gel-shift analysis of APL cells showed elevated levels of PU.1 binding activity to the M-CSF receptor promoter, particularly after ATRA stimulation. Interestingly, the rise of PU.1 binding activity as well as of PU.1 levels after ATRA treatment was significantly higher in APL patients exhibiting monocytic maturation, as compared to those that did not undergo monocytic differentiation. A variable proportion of ATRA-induced APL cells exhibited monocyte-like morphology and immunophenotype: the proportion of monocytic cells was consistently increased by combined treatment with ATRA and diverse hematopoietic growth factors cocktails, which always comprised M-CSF. Monocytic cells originating from in vitro ATRA-induced maturation of APL cells derive from the leukemic clone as suggested by two lines of evidence: (1) monocytic cells harbor the 15;17 translocation; (2) monocytic cells possess Auer bodies. The c-fms(bright) leukemic blasts preferentially showed the capacity for monocytic differentiation as compared to the c-fms(dim/-) subset: indeed, enforced expression of c-fms into NB4, a PML/RAR alpha+ cell line, favored the onset of monocytic maturation. Finally, low c-fms expression was observed in an APL relapsing patient resistant to ATRA, as well as in an APL case with t(11;17), PLZF/RAR alpha+. These observations indicate that PML/RAR alpha+ APL blasts are bipotent for differentiation through both neutrophilic and monocytic lineages, whereby monocytic differentiation is linked to c-fms expression and stimulation.

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.

The acute promyelocytic leukemia (APL)-specific t(15;17) chromosome abnormality is characterized at the molecular level by rearrangement of the PML and RAR alpha genes, resulting in fusion PML/RAR alpha mRNA and a chimeric protein. Besides its relevance in the pathogenesis of the disease, this hybrid gene represents a specific tumor marker that is rapidly detectable by reverse transcriptase-polymerase chain reaction (RT-PCR) in the RNA extracted from leukemic blasts. Several studies have highlighted the clinical relevance of PML/RAR alpha detection, which provides a specific diagnosis, prognostic information, and prediction of relapse when monitoring residual disease during the follow-up. In fact, this hybrid gene is detected in 100% of APLs. Rare cases of patients with a morphological diagnosis of FAB M3 AML who lack the specific PML/RAR alpha abnormality have been reported as being unresponsive to differentiation treatment. Finally, all the studies reported so far on PCR monitoring in APL have documented that the identification of small amounts of residual disease at remission strongly predicts impending relapse. Thus, RT-PCR of the hybrid PML/RAR alpha gene is currently performed prospectively as part of cooperative clinical trials aimed at better addressing post-remission treatment in APL.