Lenke Molnár

Detection of TNFα expression in the bone marrow and determination of TNFα production of peripheral blood mononuclear cells in myelodysplastic syndrome

TNFα is a highly active cytokine which plays an important role in the regulation of apoptotic cell death, a mechanism involved in the pathophysiology of myelodysplastic syndrome (MDS). In this study we investigated the expression of TNFα of the bone marrow trephine biopsies by immunohistochemical method and the TNFα production of peripheral blood mononuclear cells by ELISA method in 15 patients affected by MDS. Five of seven patients without excess of blasts showed high or intermediate TNFα expression in the bone marrow biopsies, whereas two patients with excess of blasts were negative and one had low expression. The five CMML patients revealed low or intermediate expression. The production of TNFα by the PBMC was analysed in 10 patients, four patients with RA and two with CMML produced higher level of TNFα which increased after stimulation with phorbol myristic acetate, but none of the RAEB patients revealed increase in TNFα production. In conclusion we suppose that increased TNFα expression and production by PBMC may be an indirect evidence of the role of increased apoptosis in low risk MDS patients.

Clinical Importance of Transforming Growth Factor-β but Not of Tumor Necrosis Factor-α Gene Polymorphisms in Patients with the Myelodysplastic Syndrome Belonging to the Refractory Anemia Subtype

Objectives: Tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) are cytokines that play key roles in the myelodysplastic syndrome (MDS). There have been several reports on the presence of genetic polymorphisms in the DNA sequence encoding the leader sequence of the TGF-β1 protein, located in codon 10 in exon 1 and in the –308 promoter region of TNF-α. The objective of this study was to investigate the association between TNF-α and TGF-β1 gene polymorphisms and the susceptibility to MDS and the progression of the disease among patients with MDS belonging to the refractory anemia (RA) subtype. Methods: The diagnosis of MDS (n = 50) was based on the FAB criteria. The TNF-α genotypes were analyzed by PCR-RFLP and the TGF-β genotypes were analyzed using an amplification refractory mutation system. Results and Conclusions: Compared with healthy control subjects, patients with RA showed no significant deviations in genotype or allele frequencies of TNF-α. The TT homozygosity at codon 10 of TGF-β1 was significantly higher among patients with bi- or pancytopenia (severe group) than in the patients with anemia only (mild group; odds ratio = 6.99, p = 0.003). These findings suggest that the TGF-β1 gene polymorphism in codon 10 and the –308 TNF-α gene polymorphism do not predispose to the development of RA, but the TGF-β1 gene polymorphism may affect disease progression.

Multiple constitutional chromosome translocations of familial nature in Philadelphia chromosome-positive chronic myeloid leukemia: a report on a unique case.

A case of Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) featuring 2 additional balanced translocations, t(2;5) and t(6;12), as well as a robertsonian translocation, t(13;14), was diagnosed by routine bone marrow karyotyping. The breakpoints did not involve previously described CML-related chromosomal regions in any of the 3 translocations. Despite the patient’s partial response following imatinib therapy, all Ph- bone marrow metaphases persistently had the 3 additional chromosomal changes. Moreover, stimulated peripheral B-lymphocytes from the patient also showed the same chromosomal changes, suggesting that we had found a complex constitutional chromosome aberration unrelated to the leukemia. Peripheral blood karyotype analyses of 6 of the 7 closest relatives from 3 generations demonstrated at least 1 of these aberrations, although in different combinations. Standard bone marrow or peripheral blood karyotyping of hematologic disorders may uncover otherwise symptomless, unrelated constitutional changes together with disease-specific chromosome aberrations. A triple constitutional chromosome aberration combined with a hematologic disorder has not been described until now. In addition, multiple constitutional aberrations persisting through at least 3 generations seem to be extremely rare. At present, no direct evidence exists to support a causative relationship between the familial translocations and leukemogenesis.

Silent Philadelphia Chromosome: A Distinct Developmental Stage in a Philadelphia Chromosome-Positive Chronic Myeloproliferation?

In this paper, a patient is described who presented with peripheral blood and bone marrow features uncharacteristic of chronic granulocytic leukemia, which proved to be Philadelphia (Ph) chromosome-positive by metaphase and interphase cytogenetic analyses but lacked the p210 type of BCR/ABL fusion gene mRNA product by two different sensitive RT-PCR assays. In the course of the 32-month follow-up with a termination into a myeloblastic crisis, molecular investigations were performed four times. They indicated a constantly high rate of Ph positive cells and lack of BCR/ABL mRNA expression, except in the second investigation, when the patient showed reverse transcription polymerase chain reaction positivity with b3/a2 type of chimera, fusion gene mRNA expression, and a striking change in the bone marrow histology. Our findings might indicate that the dormant Ph chromosome state may exist not only at the primitive progenitor, but also at the entire peripheral blood cell compartment level.

Lineage-specific clonality analysis of chronic myeloproliferative disorders and myelodysplastic syndrome by human androgen receptor assay.

In myelodysplastic syndrome (MDS) as well as chronic myeloproliferative disorders (CMPD) others than chronic myeloid leukemia the frequency of pathognomonic genetic aberrations is very low and, therefore, X chromosome inactivation (XI) assays may help in assessing the clonality. To establish specific clonality criteria on XI, human androgen receptor assay (HUMARA) was performed on sorted myeloid and lymphoid peripheral blood cells of 21 healthy females. Clonality criteria 1 and 2 conferring at least 90% specificity were set based on the ranges and differences of XI number (XIN) describing the ratio of representation of the two alleles in as well as in between reactive myeloid and lymphoid compartments. Spiking experiments indicated that the test identifies clonality reliably when no more than 40–50% reactive cells are admixed. In the CMPD and MDS cases peripheral myeloid cells were monoclonal by one of the two criteria in 71–100%, whereas lymphoid cells in 28–75%. The results of HUMARA, available in 73% of the female patients, supported the clinicopathological data in 84% as well as proved pluripotent stem cell origin in 31–75% and 21% of CMPDs and MDS, respectively.