Donor Selection Based on Killer Cell Immunoglobulin-Like Receptor (KIR) Genotype May Improve Outcome After T-Cell-Replete Haploidentical Transplantation.

BACKGROUND: In haploidentical stem cell transplantation (SCT), the "ideal donor" selection is not performed in a standardized way according to killer cell immunoglobulin-like receptor (KIR) genotype expressed by potential donors. The aim of this study was to evaluate the relevance of KIR genotype in a series of patients submitted to haploidentical SCT in our center. METHODS: We retrospectively analyzed 30 patients that were prepared with the use of a conditioning regimen including thiotepa-busulfan-fludarabine with high doses of post-transplantation cyclophosphamide (CyPT) and tacrolimus as graft-versus-host disease (GVHD) prophylaxis. We analyzed the impact of the KIR genotype variables (donor AA/Bx haplotype, donor B content, KIR inhibitor mismatches, and mismatching in KIR ligands in the graft-versus-host direction and the host-versus-graft direction) on overall survival, GVHD-free survival, and event-free survival. RESULTS: Statistical significance was found for the presence of mismatches on KIR ligands in the graft-versus-host direction in relation to the diagnosis of chronic GVHD (54% vs 100%; P = .004). Significance was also found for the effect of the donor presence AA or Bx haplotype in relation to the diagnosis of chronic GVHD (50% vs 86%; P = .033). CONCLUSIONS: KIR genotyping can provide useful information that can help us with the right donor choice for haploidentical SCT without T-cell depletion with high doses of CyPT. Donors with Bx haplotype that do not show KIR ligand incompatibilities in the graft-versus-host direction may provide a lower risk of GVHD.

Comparative study of BCR-ABL1 quantification: Xpert assay, a feasible solution to standardization concerns.

The level of BCR-ABL1 reached after treatment with tyrosine kinase inhibitors is an effective marker of the therapeutic response and a good survival predictor in chronic myeloid leukemia (CML) patients. However, no agreement has yet been achieved about either the standardization of the technique to determine BCR-ABL1 or the interpretation of the results. The aim of this study was to compare the method currently recommended by the European Leukemia Net, which includes the application of a conversion factor to express the results in international scale, with an automated method (Xpert BCR-ABL™, Cepheid). BCR-ABL1 transcript quantification was performed in 117 samples from CML patients in two different laboratories by both methods, and the results were compared by statistical procedures. A high linear correlation was obtained in the results between the two methods. The concordance at logarithmic intervals reached 62 %. When the major molecular response (MMR) was analyzed, 85 % agreement was achieved. The automated method provides reproducible results and does not show significant differences compared with the traditional method. As a clinical tool, Xpert correctly classified the patients in MMR and can be considered a useful alternative for the molecular follow-up of CML patients.

ASPP1, a common activator of TP53, is inactivated by aberrant methylation of its promoter in acute lymphoblastic leukemia.

We have analyzed the regulation and expression of ASPP members, genes implicated in the regulation of the apoptotic function of the TP53 tumor-suppressor gene, in acute lymphoblastic leukemia (ALL). Expression of ASPP1 was significantly reduced in ALL and was dependent on hypermethylation of the ASPP1 gene promoter. Abnormal ASPP1 expression was associated with normal function of the tumor-suppressor gene TP53 in ALL. The analyses of 180 patients with ALL at diagnosis showed that the ASPP1 promoter was hypermethylated in 25% of cases with decreased mRNA expression. Methylation was significantly higher in adult ALL vs childhood ALL (32 vs 17%, P = 0.03) and T-ALL vs B-ALL (50 vs 9%, P = 0.001). Relapse rate (62 vs 44%, P = 0.05) and mortality (59 vs 43%, P = 0.05) were significantly higher in patients with methylated ASPP1. DFS and OS were 32.8 and 33.7% for patients with unmethylated ASPP1 and 6.1 and 9.9% for methylated patients (P < 0.001 y P < 0.02, respectively). On the multivariate analysis, methylation of the ASPP1 gene promoter was an independent poor prognosis factor in ALL patients. Our results demonstrate that decreased expression of ASPP1 in patients with ALL is due to an abnormal methylation of its promoter and is associated with a poor prognosis.

Reliable quantification of hematopoietic chimerism after allogeneic transplantation for acute leukemia using amplification by real-time PCR of null alleles and insertion/deletion polymorphisms.

Increasing mixed chimerism (MC) after allogeneic stem cell transplantation (SCT) has been associated with a high risk of relapse in acute leukemia. We evaluated a new method for chimerism detection, based on the quantitative real-time PCR (qrt-PCR) amplification of null alleles or insertion/deletion polymorphisms (indels). All qrt-PCR assays with null alleles and indels attained a sensitivity of at least 10(-4), as well as good intra- and interassay concordance, and a high accuracy in experiments with cell mixtures. Informativeness was found in 80.3% of the 61 donor/recipient pairs tested. Nonrelapsed patients showed a progressive decrease in peripheral blood chimerism to values below 0.01% (complete chimerism (CC)). Bone marrow chimerism failed to reach CC more than 4 years after SCT. Increasing MC was observed prior to relapse in 88.2% of patients. Compared with conventional PCR amplification of variable number of tandem repeats, qrt-PCR predicted a significantly higher number of relapses (88.2 vs 44.4%) with a median anticipation period of 58 days. In conclusion, chimerism determination by qrt-PCR amplification of null alleles and indels constitutes a useful tool for the follow-up of patients with acute leukemia after SCT, showing better results than those obtained with conventional PCR.

Transcriptional silencing of the Dickkopfs-3 (Dkk-3) gene by CpG hypermethylation in acute lymphoblastic leukaemia.

DKK-3: is a newly characterised mortalisation-related gene and an antagonist of the Wnt oncogenic signalling pathway whose expression is decreased in a variety of cancer cell lines, suggesting that the Dkk-3 gene, located at chromosome 11p15.1, functions as a tumour suppressor gene. Although 11p15 is a 'hot spot' for methylation in acute lymphoblastic leukaemia (ALL), the role of Dkk-3 abnormalities has never been evaluated in this disease. We analysed CpG island methylation of the Dkk-3 promoter in six ALL cell lines and 183 ALL patients. We observed Dkk-3 hypermethylation in all cell lines and in cells from 33% (60/183) of ALL patients. Moreover, Dkk-3 methylation was associated with decreased Dkk-3 mRNA expression and this expression was restored after exposure to the demethylating agent 5-AzaC. Clinical features did not differ between hypermethylated and unmethylated patients. Estimated disease-free survival (DFS) and overall survival at 10 and 11 years, respectively, were 49.8 and 45.6% for normal patients and 10.5 and 15.1% for hypermethylated patients (P=0.001 and 0.09). Multivariate analysis demonstrated that Dkk-3 methylation was an independent prognostic factor predicting DFS (P=0.0009). Our data suggest that Dkk-3 methylation occurs at an early stage in ALL pathogenesis and probably influences the clinical behaviour of the disease.