Summary. In this study, we used multiparameter flow cytometry to quantify minimal residual disease (MRD) in 165 serial bone marrow samples from 40 patients diagnosed with acute lymphoblastic leukaemia (ALL) who underwent allogeneic stem cell transplantation (allo-SCT) from siblings (n ¼ 34) or unrelated donors (n ¼ 6). Samples were prospectively taken from 24 patients before starting the conditioning regimen, at days +30, +60 and +90 and subsequently every 2-3 months. Samples from 16 patients in complete remission (CR) after allo-SCT were taken at least twice. Six of 24 patients harboured MRD (0AE2-10% of mononuclear cells) at transplant and 18 were negative. Estimated disease-free survival for the MRD+ and MRDgroups at transplant was 33AE3% and 73AE5% respectively (P ¼ 0AE03). During follow-up, increasing MRD levels were detected in nine patients, a finding that preceded marrow relapse by 1-6 months. Two patients with stable low MRD levels remained in CR. When we used flow cytometry to test the effect of donor leucocyte infusions (DLI) in six patients, we observed that the only sustained remission was achieved when DLI was applied prior to overt relapse. We conclude that MRD by flow cytometry can rapidly assess tumoral burden before transplant to predict outcome, and can be clinically useful for the timing of DLI for increasing levels of leukaemia after transplant.

Persistent thrombocytopenia is a common complication after allogeneic hematopoietic stem cell transplantation (allo-SCT). Romiplostim and eltrombopag are the currently available thrombopoietin receptor agonists (TPO-RAs), and some studies with very small numbers of cases have reported their potential efficacy in the allo-SCT setting. The present retrospective study evaluated the safety and efficacy of TPO-RAs in 86 patients with persistent thrombocytopenia after allo-HSCT. Sixteen patients (19%) had isolated thrombocytopenia (PT), and 71 (82%) had secondary failure of platelet recovery (SFPR). TPO-RA therapy was started at a median of 127 days (range, 27 to 1177 days) after allo-SCT. The median initial and maximum administered doses were 50 mg/day (range, 25 to 150 mg/ day) and 75 mg/day (range, 25 to 150 mg/day), respectively, for eltrombopag and 1 mg/kg (range, 1 to 7 mg/kg) and 5 mg/kg (range, 1 to 10 mg/kg), respectively, for romiplostin. The median platelet count before initiation of TPO-RA therapy was 14,000/mL (range, 1000 to 57,000/mL). Platelet recovery to 50,000/mL without transfusion support was achieved in 72% of patients at a median time of 66 days (range, 2 to 247 days). Eighty-one percent of the patients had a decreased number of megakaryocytes before treatment, showing a slower response to therapy (P = .011). The median duration of treatment was 62 days (range, 7 to 700 days). Grade 3-4 adverse events (hepatic and asthenia) were observed in only 2% of the patients. At last follow-up, 81% of patients had discontinued TPO-RAs and maintained response, and 71% were alive. To our knowledge, this is the largest series analyzing the use of TPO-RAs after allo-SCT reported to date. Our results support the efficacy and safety in this new setting. Further prospective trials are needed to increase the level of evidence and to identify predictors of response.

ex vivo expansion protocols, OX40 blocking might be crucial to optimize the use of Treg to prevent GVHD.

Wnt5a is a member of the Wnt family of proteins that signals through the non-canonical Wnt ⁄ Ca 2+ pathway to suppress cyclin D1. Deregulation of this pathway has been found in animal models suggesting that it acts as tumour suppressor in acute myeloid leukemia (AML). Although DNA methylation is the main mechanism of regulation of the canonical Wnt pathway in AML, the role of WNT5A abnormalities has never been evaluated in this clinical setting. The methylation status of WNT5A promoter-exon 1 was analyzed by methylation-specific PCR and sequencing in eleven AML-derived cell lines and 252 AML patients. We observed WNT5A hypermethylation in seven cell lines and in 43% (107 ⁄ 252) of AML patients. WNT5A methylation was associated with decreased WNT5A expression (P < 0.001) that was restored after exposure to 5-Aza-2'-deoxycytidine. Moreover, WNT5A hypermethylation correlated with upregulation of CYCLIN D1 expression (P < 0.001). Relapse (15% vs 37%, P < 0.001) and mortality (61% vs 79%, P = 0.004) rates were lower for patients in the non-methylated group. Disease-free survival and overall survival at 6 and 7 years, respectively, were 60% and 27% for unmethylated patients and 20% and 0% for hypermethylated patients (P = 0.0001 and P = 0.04, respectively). Interestingly, significant differences were also observed when the analysis was carried out according to cytogenetic risk groups. We demonstrate that WNT5A, a putative tumor suppressor gene in AML, is silenced by methylation in this disease and that this epigenetic event is associated with upregulation of CYCLIN D1 expression and confers poor prognosis in patients with AML. (Cancer Sci 2010; 101: 425-432) W nt proteins form a large family of secreted glycoproteins that activate the highly conserved Wnt pathway through their receptor Frizzled. Wnt-Frizzled signaling controls a wide variety of processes, including proliferation, differentiation, migration, and cell-cell adhesion, playing a crucial role not only during development but also in hematopoietic stem cell maintenance and oncogenesis.(1-4) Wnt signaling occurs through the canonical and non-canonical pathways. The canonical pathway has a central mediator, b-catenin. In the absence of Wnt ligand, b-catenin is phosphorylated and ubiquitinated by the activity of a multiprotein destruction complex that contains axin, adenomatous poliposis coli, glycogen-synthase kinase 3b, and casein kinase 1, leading to proteasome-mediated degradation. On the other hand, binding of Wnt to the Frizzled receptor leads to inhibition of the destruction complex through activation of the protein Dishevelled. Then, b-catenin is stabilized and translocated into the nucleus. This allows binding of b-catenin to transcription factors such as lymphoid enhancing factor-1 and T-cell factor, and leads to transcriptional activation of multiple target genes like CYCLIN D1 and C-MYC.(1-4)The non-canonical signaling pathway, although not completely understood, is independent of b-catenin and includes two defined pathways: the planar cell polari...