Jean-Luc Vaerman

Early immunological monitoring after pediatric liver transplantation: cytokine immune deviation and graft acceptance in 40 recipients.

Cytokine deviation may be a factor contributing to graft acceptance. We analyze, in the context of liver transplantation, circulating cytokine levels and their mRNA precursors in liver biopsy samples to study a putative correlation with early immunologic outcome. Forty primary pediatric liver recipients were submitted to a prospective immune monitoring protocol, including 8 of 40 patients with an early, biopsy‐proven acute rejection episode. The 32 patients with graft acceptance showed markedly increased interleukin (IL)‐10 blood levels at 2 hours after reperfusion on days 1 and 4 after transplantation as compared with baseline, whereas patients with graft rejection only exhibited increased IL‐10 levels at 2 hours. A good correlation was observed between IL‐10 peripheral levels and levels ascertained by IL‐10 reverse transcriptase–polymerase chain reaction at 2 hours and on day 7. Patients with graft acceptance also showed a decrease in interferon gamma (IFN‐γ) at 1 and 2 hours after reperfusion on days 1, 4, 7, 14, and 28 after transplantation. One patient with graft tolerance who had subsequent immunosuppression withdrawal after posttransplantation lymphoproliferative disease showed a similar intraoperative IL‐10 pattern, whereas posttransplantation tumor necrosis factor alpha and IFN‐γ levels greatly decreased. The occurrence of cytokine immune deviation may therefore be related to early graft acceptance in children who receive liver transplants. Liver Transpl 13:426–433, 2007.

Chronic Myeloid Leukemia with a Rare Variant Philadelphia Translocation: t(9;22;21)(q34;q11;q22)

A case of chronic myeloid leukemia displaying an uncommon t(21;22)(q22;q11) is reported. For the first time, this translocation has been characterized by fluorescence in situ hybridization (FISH) and the reverse transcriptase polymerase chain reaction (RT-PCR). FISH, with the use of whole-chromosome painting probes and probes specific for the BCR and ABL genes, showed a three-way variant Philadelphia translocation (9;22;21)(q34;q11;q22) with a BCR/ABL fusion residing on the der(22). In addition, RT-PCR demonstrated a b2a3 BCR/ABL fusion transcript. Underlying mechanisms and prognostic implications are discussed.

Beta-thalassaemia in indigenous Belgian families: identification of a novel mutation

Abstract The molecular basis of β-thalassemia was investigated at the DNA level in 28 Belgians from 14 unrelated families. All the patients were heterozygous for β-thalassaemia. Seven different mutations were identified using a combination of dot-blot hybridization with allele-specific oligonucleotide probes and direct automated fluorescence-based DNA sequencing. Among these mutations, four are commonly found in the Mediterraneans – codon 8 (–AA), IVS-I-1 (G→A), IVS-I-6 (T→C) and codon 39 (C→T) – and two have occasionally been reported – initiation codon (T→C) and codon 35 (C→A). The last mutation, a –CC deletion at codons 38/39, appears to be a novel mutation and can routinely be investigated by AvaII restriction on amplified DNA. We report our findings, discuss the diversity of the mutations found in Belgium and show the usefulness of direct DNA sequencing in a population in which the molecular defects of β-thalassaemia have yet to be characterized and in which screening is hampered by the wide range of potential mutations.

Expression of lineage markers using real-time quantitative polymerase chain reaction (RT-qPCR) in normal and in leukemia bone marrow

Abstract Background: The study of lineage markers by real-time quantitative polymerase chain reaction (RT-qPCR) at diagnosis enables differentiation between acute myeloblastic leukemia, B- or T-lineage acute lymphoblastic leukemia, without cell sorting. Our objective was to assess the relationship between protein expression and the amount of lineage marker mRNA in acute leukemia samples and to determine whether four lineage markers could be used to differentiate between normal and acute leukemia bone marrow (BM) without cell sorting. Methods: Quantification of the mRNA of CD19, CD79a, CD3e, and myeloperoxidase was performed by RT-qPCR on 130 acute leukemia BM samples at diagnosis and on 20 BM samples from healthy donors, without cell sorting. Immunophenotyping of leukemia samples was performed after manual gating around the blastic population. Results: Reference values for the four lineage markers were established by RT-qPCR for normal BM. The mRNA expression levels of these four lineage markers allowed the distinction between normal samples and 100% of acute leukemia samples. Conclusions: With 92% congruence for protein expression and amount of mRNA in acute leukemias, these four lineage markers, essential for diagnosis and subclassification of acute leukemias by flow cytometry, also represent excellent candidate genes when using RT-qPCR technology as a diagnostic tool for molecular cancer class prediction. Clin Chem Lab Med 2009;47:419–26.