ALBERT

Beschreibung: Abstract 1329 Poster Board I-351 The detection of minimal residual disease (MRD) in AML patients after allogeneic stem cell transplantation (SCT) is important for the early detection of relapse and the selection of patients who can benefit from immunomodulation. Up to 65% of patients with AML lack of molecular markers useful for follow-up. The Wims tumor gene (WT1) is overexpressed in more than 80% of AML patients at diagnosis, hence its utility as MRD marker in these patients. The objective of this study is to analyze the usefulness of WT1 expression for MRD study and relapse prediction in patients with AML after allogeneic SCT and to compare the results with those from flow cytometry and chimerism. Methods 149 samples were studied (63 BM and 86 PB) from 11 patients with AML (table 1) lacking of other molecular markers at diagnosis, who underwent allogeneic SCT in a single center from 2003 and 2008. Quantification of WT1 expression was performed by quantitative RT-PCR (LightCycler 1.2) using cDNA obtained from 1 ug of RNA (Trizol®, Invitrogen). K562 cell line was used as calibrator and ABL as reference genes (relative quantification). Cut-off values were defined at 0.5% in BM samples and 0.01% in PB. Results were correlated with those from flow cytometry and chimerism from the same samples. Results WT1 overexpression correlated with the presence of disease before transplantation (table 1). Post-transplantation, 5 of the 11 patients showed low and stable WT1 levels in BM and PB in the follow-up, remaining in CR (median follow-up 48 months, range 8-61). These 5 patients showed complete chimerism (CC) and negative MRD by flow cytomety (figure A). On the other hand, 6 from the 11 patients showed WT1 overexpression after SCT: 4 of them showed after initial low values, a significant increase in WT1 expression up to reaching positive levels in a median of 139 days (50-167), while 2 patients showed positive values continuously. Two patients showed positive levels only in PB samples, one only in BM and 3 both in BM and PB. From these 6 patients, 5 relapsed (1 with extramedullar disease who showed WT1 overexpression only in PB) in a median of 9 months after SCT (2.4-11). In one case, WT1 overexpression occured at the same time as relapse, while in the other 4 cases, relapse occured in a median of 137 days (134-170) after a significant increase in WT1 levels was seen in two consecutive samples. In these 5 relapsed patients, neither flow cytometry nor chimerism predicted relapse (figure B). Conclusions WT1 overexpression correlated with disease burden in AML patients after allogeneic SCT. In realapsed patients, relapse both medullar and extramedullar was anticipated sinificantly by WT1 overexpression compared to flow cytometry and chimerism. Quantification of WT1 overexpression by RT-PCR should be used for MRD detection in the follow-up after SCT in AML patients (not only in BM but also in PB for extramedullar disease detection) in order to facilitate immunossupresive therapy management and to select early DLI candidates. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Background: Polymorphisms in regulatory sequences of cytokine genes are known to influence their expression and, therefore, the intensity of the immune response. Some of such polymorphisms have been associated with the outcome of stem cell transplantation (SCT), which supports the hypothesis of a genetic predisposition towards certain complications post-transplants. Objective: To evaluate the association between donor (D) and recipient (R) genotype in the IL-6 gene -174G/C SNP polymorphism with the development of complete or mixed chimerism (MC) and, consequently, with complications such as graft rejection, graft versus host disease (GVHD) and survival after SCT. Patients and methods: The study included 16 SCT (9 ablative, 3 T cell depleted, 4 reduced intensity conditioning). The IL-6 -174G/C SNP genotypes were determined by PCR on genomic DNA from Rs and Ds, using allele-specific oligonucleotides (ASO-PCR) as primers (forward: 5′-ccctagttgtgtcttgcc-3′ or 5-′ccctagttgtgtcttgcg-3′; reverse: 5′-gagcttctctttcgttcc-3′). Logistic regression was applied to identify associations between the polymorphism, the development of MC and of the mentioned complications. The reduced number of SCT analyzed, precluded the use of Pearson’s and/or Fisher’s p values or further statistics. Results: The frequency of the different genotypes was 44.4% CC, 33.3% GC and 22.2% GG for Rs, and 33.3% CC, 44.4% GC and 22.2% GG for Ds. 88.9% D/R pairs were matched for the polymorphism and 11,1% mismatched. None of the patients transplanted from matched donors showed MC at any time post-SCT while all mismatched patients developed MC (Table 1). MC in the D/R mismatched group was associated with a greater rate of graft rejection and with a reduced incidence of acute GVHD (aGVHD) when compared with the D/R matched group (Table 1). No association was observed in this sample between the polymorphism analyzed and the development of chronic GVHD (cGVHD). Although graft rejection was early diagnosed and successfully treated with immunosuppression withdrawal and donor leukocyte infusion, the D/R matched group showed higher mortality rate that the mismatched group (Table 1). Conclusions: The present study suggests an association between D/R matching for the IL-6 gene -174G/C SNP polymorphism and the development of MC and, consequently, of different complications after SCT. The analysis of a larger number of patients will eventually allow to confirm these observations as well as to establish this type of studies as a means for an improved management of transplanted patients. Table 1 IL6 -174G/C MC rejection aGVHD cGVHD Exitus D/R matched 0% 0% 75% 87.5% 50% D/R mismatched 100% 100% 0% 100% 100%

Beschreibung: WHO criteria defines platelet counts above 600×109/L as the threshold for essential thrombocythemia (ET) diagnosis. It has been argued that such threshold excludes a number of patients with ET with platelet counts below 600×109/L. Recently, a proposal for revision of the World Health Organization (WHO) diagnostic criteria for ET has been published, which includes the combination of histological bone marrow study and testing of JAK2 mutation. Design and methods: Retrospective analysis of 92 patients with ET diagnosis between 1989 and February 2008, isolating the subgroup of patients with platelet counts below 600×109/L. The aim of this study was to analyze the applicability of the 2008 WHO criteria in this subgroup. Results: Of the 92 patients, 30 patients did not fulfill the WHO criteria due to platelet counts