When can real-time quantitative RT-PCR effectively define molecular relapse in acute promyelocytic leukemia patients? (Results of the French Belgian Swiss APL Group).

10-20% of APL patients relapse and the challenge remains to early identify these patients to improve survival rate. We report PML-RARalpha transcript detection by RQ-PCR in 260 consecutive APL patients (n = 970 samples). 223 patients with samples of sufficient RNA quality to demonstrate they reached molecular remission were monitored for MRD. During follow-up, 38 of these patients were tested positive for PML-RARalpha mRNA. 13 out of the 38 patients (34%) effectively developed hematological relapse. In the first positive sample, specific PML-RARalpha NCN thresholds over which, or under which, patients could effectively be predicted to relapse or not, were identified and subsequently validated in a second cohort.

Favourable outcome in an APL patient with PLZF/RARalpha fusion gene: quantitative real-time RT-PCR confirms molecular response.

Rare cases of acute promyelocytic leukemia (APL) are associated with a t(11;17) translocation and a PLZF-RARalpha fusion transcript. Because of molecular specificities of the fusion protein, ATRA efficiency is often reduced in these cases. We present herein the case of an 83-year old patient which has been successfully treated by ATRA and Daunorubicin. The described quantitative RT-PCR method allowed successful monitoring and confirmation of the molecular response.

Frequent antibody production against RARalpha in both APL mice and patients.

In an acute promyelocytic leukemia (APL)-transplantable mouse model, we previously reported the presence of antibodies recognizing PML-RARalpha and RARalpha in the sera of ATRA-treated mice. To evaluate this immune response, we determined the prevalence of anti-RARalpha antibodies in a cohort of 48 APL mice, treated by ATRA (n = 24) or by placebo pellets (n = 24), and in a preliminary subset of 9 patients with APL using a specific enzyme-linked immunosorbent assay (ELISA). In APL mice, significantly higher antibody levels were observed at the latest time points (day 48 to 58 levels superior to day 15 to 18 or day 28 to 38 levels). Antibody levels were higher in ATRA-treated mice than in placebo-treated mice and were also predictive of better survival. In the patients with APL, anti-RARalpha antibodies were detected at diagnosis and after maintenance therapy, reminiscent of the ATRA-treated APL mice. Antinuclear or antineutrophil cytoplasmic autoantibodies were also detected. These data reveal for the first time that in patients with APL an immune response may be detected at diagnosis and enhanced after maintenance therapy.

Cooperative action of 1alpha,25-dihydroxyvitamin D3 and retinoic acid in NB4 acute promyelocytic leukemia cell differentiation is transcriptionally controlled.

All-trans-retinoic acid (RA) and 1alpha,25-dihydroxyvitamin D3 (1,25D3) are involved in the control of hematopoiesis and have been suggested to play a role in cellular differentiation and are as such potent inducers of differentiation of myeloid leukemia cells. In this study, we show that, in promyelocytic NB4 cells, addition of 1,25D3 enhances terminal granulocytic RA-dependent differentiation concomitant with the enhanced activation of the RA transcriptional activity through an RARbeta promoter. By EMSA and ChIP assays, we further demonstrate that, while both VDR and RAR are bound to the RARbeta promoter in NB4 cells, addition of 1,25D3 increases VDR binding to this promoter, while that of RA induces the release of VDR and increases the binding of RAR. Thus, contrary to normal myeloid cells, 1,25D3 does not act as a transrepressor of RA transcriptional activity in leukemic cells, suggesting that transcriptional regulation of RA-target genes may be modified in malignant cells. In promyelocytic leukemic cells, the combination of 1,25D3 and RA results in both enhanced transactivation and differentiation.

1 alpha,25-dihydroxyvitamin D3 transrepresses retinoic acid transcriptional activity via vitamin D receptor in myeloid cells.

Granulocytes and monocytes originate from a common committed progenitor cell. Commitment to either granulocytic or monocytic lineage is triggered by specific extracellular signals involving cytokines or nuclear receptor ligands (all-trans-retinoic acid (RA) and 1 alpha,25-dihydroxyvitamin D(3)). Here we show that the stimulatory effect of 1 alpha,25-dihydroxyvitamin D(3) on the production of monocytic colonies (CFU-M) is accompanied by a repression of granulocytic colony (CFU-G) production. We further demonstrate that in bipotent HL-60 myeloid cells as in purified human CD34+ myeloid progenitor cells, the 1 alpha,25-dihydroxyvitamin D(3)-induced monocytic differentiation is concomitant with a direct inhibition of the RA-transcriptional activity. Indeed, a transrepression of the RAR beta RA-target gene promoter via formation of a nuclear complex involving VDR was identified in vitro and in vivo. The fact that binding of RXR-RAR on DR3 is not observed suggests that contrary to RA-induced granulocytic differentiation, 1 alpha,25-dihydroxyvitamin D(3)-mediated monocytic differentiation requires positive and negative transcriptional controls both likely mediated by the RXR-VDR heterodimer. These novel findings implicate that 1 alpha,25-dihydroxyvitamin D(3) exerts a dominant negative effect on the RA-dependent granulocytic commitment of human bone marrow cells via repression of the RA-target gene promoters. Hence, the transcriptional response to RA and 1 alpha,25-dihydroxyvitamin D(3) in myeloid cells depends on a complex combinatory pattern of interaction among different nuclear receptors with DNA.