Molecular and flow cytometry characterization during the follow-up of three simultaneous lymphoproliferative disorders: hairy cell leukemia, monoclonal B-cell lymphocytosis, and CD4(++) /CD8(+/- dim) T-large granular lymphocytosis--a case report.

The simultaneous diagnosis of hairy cell leukemia and monoclonal B-cell lymphocytosis with the characteristics of "indolent" chronic lymphocytic leukemia is rare but not unknown. However, an association with a third clonal lymphoproliferative disorder has not previously been described. We report the simultaneous presence of hairy cell leukemia, monoclonal B-cell lymphocytosis, and alpha beta CD4(++) /CD8(+) T-cell large granular lymphocytosis in a 63-year-old man. After the diagnosis, the three lymphoproliferative disorders (i.e., two of B-cell lineage and one of T-cell lineage) were characterized by analysis of multiple sequential bone marrow and peripheral blood samples using flow cytometry and molecular techniques. We discuss these findings in the context of chronic antigen stimulation, immunosuppression, and apoptotic pathway alterations, which might be implicated in the accumulation of these abnormal clones in the same patient. Because the phenotype of the three clones is compatible with fully differentiated B lymphocytes (consistent with a postgerminal origin) and T-CD4(++) cells, we favor the possibility of an antigen-driven mechanism and a dysregulation of homeostatic apoptosis in this patient.

Are myeloma patients with renal failure candidates for autologous stem cell transplantation?

INTRODUCTION: Renal function is one of the most important prognostic factors in multiple myeloma (MM). Patients with renal failure are generally excluded from high dose therapy even though they display a poor prognosis with conventional chemotherapy schemes. The aim of this study was to analyze the outcome of MM patients with renal insufficiency undergoing autologous stem cell transplantation (ASCT), including the evaluation of the quality of PB stem cell collections, kinetics of engraftment, transplant-related mortality, response to high dose chemotherapy and survival. MATERIALS AND METHODS: From a total of 566 valuable patients included in the MM Spanish ASCT registry, three groups of patients were defined: group BA, patients with abnormal renal function at diagnosis but normal at transplant (73 cases); group BB, patients with abnormal function both at diagnosis and at transplant (14 cases); and group AA (control group, 479 cases), patients who constantly had normal renal function. RESULTS AND CONCLUSION: Patients from groups BA and BB presented with a significantly higher number of adverse prognostic factors, reflecting that we were dealing with high tumor MM cases, as compared with patients from group AA. The number of mononuclear cells, CD34+ cells and CFU-GM cells collected in patients with non-reversible renal insufficiency was similar to those harvested in MM patients with normal renal function. Moreover, neutrophil and platelet engraftments were identical in patients with and without renal failure (days +11 and +12, respectively). By contrast, transplant-related mortality (TRM) was significantly higher in group BB patients (29%) than in groups BA (4.1%) and AA (3.3%). In multivariate analysis only three variables showed independent influence on TRM: poor performance status (ECOG 3), hemoglobin <9.5 g/dl and serum creatinine > or =5 mg/dl. The response to high dose therapy was independent of renal function. Interestingly, 43% of patients from group BB showed an improvement in renal function (creatinine < 2 mg/dl) after transplant. The three-year overall survival from transplantation was 56, 49 and 61% for the BB, BA and AA groups, respectively, with a statistically significant difference favoring group AA (P<0.01). PFS did not differ significantly between the three groups of patients. In multivariate analysis the only unfavorable independent prognostic factors for overall survival were poor performance status either at diagnosis or at transplant, high beta(2)-microglobulin levels, and no response to transplant. According to these results, ASCT is an attractive alternative for MM patients with renal insufficiency, and it should not constitute a criterion for exclusion from transplant unless patients display poor performance status and very high creatinine levels (>5 mg/dl).

Coexistence of tetrasomy 8 and trisomy 8 in a case with myeloid metaplasia with myelofibrosis.

We present a case of myeloid metaplasia with myelofibrosis (MM/MF), with tetrasomy 8 as the sole cytogenetic abnormality detected by conventional cytogenetic studies. Tetrasomy 8 was also detected by in situ interphase studies and confirmed by chromosome painting in metaphase. To our knowledge, this is the first case of MM/MF with tetrasomy 8. Noteworthy is the association with neurofibromatosis.

Use of rhG-CSF in the collection of peripheral blood stem cells for autologous blood stem cell transplantation in acute myeloblastic leukemia.

The use of recombinant granulocyte colony-stimulating factor (rhG-CSF) alone is attractive for the collection of peripheral blood stem cells (PBSC) in several malignancies. In acute myeloblastic leukemia (AML), not enough experience has been gained with rhG-CSF and leukapheresis is more common after recovery from antileukemia chemotherapy. This is the case report of a patient who received rhG-CSF alone to mobilize stem cells where the cells collected in the leukapheresis bag had a blastic immunophenotype.

Hb Fannin-Lubbock in five Spanish families is characterized by two mutations: beta 111 GTC-->CTC (Val-->Leu) and beta 119 GGC-->GAC (Gly-->Asp).

We have sequenced the amplified beta-globin genes of five, apparently unrelated, Spanish adults with a fast-moving hemoglobin variant, and observed a GGC-->GAC mutation at codon 119 which identified the abnormality as Hb Fannin-Lubbock or alpha 2 beta (2)119(GH2)Gly-->Asp. In addition, we found a GTC-->CTC change at codon 111 which leads to a Val-->Leu replacement at this location. Protein analysis of the beta A and beta X chains from one of these individuals confirmed that both mutations are located on the same chromosome. It is hypothesized that some other known variants may carry an additional mutation in one of their exons, resulting in a silent amino acid substitution which may have an effect on some physicochemical property. In the case of Hb Fannin-Lubbock, it appears likely that the Val-->Leu replacement at beta 111, rather than the Gly-->Asp replacement of beta 119, is the cause of the instability of the variant. The Hb Fannin-Lubbock variant in these Spanish families had a normal oxygen affinity.

[Frequency of molecular alterations in heterozygous beta-thalassemia in southern Spain and their relation to the hematologic phenotype]. / Frecuencia de las alteraciones moleculares de la beta-talasemia heterocigota en el sur de España y su relación con el fenotipo hematológico.

INTRODUCTION: Heterozygous beta-thalassemia manifests hematologically with microcytosis, reduced red blood cell hemoglobin concentration and high hemoglobin A2 levels. Almost all molecular alterations are due to point mutations. We attempt to determinate the frequency of that mutations in the Oriental Andalusia Area, and its relationship with the hematological phenotype. PATIENTS AND METHODS: We have studied 45 heterozygous patients. DNA samples were amplified by PCR, using the printers CD7 and HI1. A 16 Kb fragment corresponding to beta globin gene was obtained and analyzed by Dot Blot assay and hybridized with allelic specific oligonucleotide (ASO) probes to detect the 6 more frequent mutations found in the South of Spain. RESULTS: Codon 39 nonsense mutation (31.1%) was the most frequent finding followed by IVS-1 NT 110 (26.7%). The relationship between hematological parameters and molecular mutations concluded that IVS-I NT 6 mutation developed a minimal anemia. DISCUSSION: From the practical point of view, this study indicates that we were able to detect more than 90% of heterozygous beta-tal. with 5 out of 6 ASO probes used in this work. Thus, our data also provides a further implication in prenatal diagnosis.

[Hemoglobin Setif (alpha 94 (G1) Asp----Tyr) in a Spanish family]. / Hemoglobina Setif (alpha 94 (G1) Asp----Tyr) en una familia española.

A family was studied who carried a slow mobility haemoglobin on cellulose acetate electrophoresis at pH 8.6. The structural analysis of the anomalous globin chain showed substitution of residual aspartic acid in position 94 of the alpha chain by tyrosine (Hb Setif). This mutation induces low oxygen affinity in the haemoglobin molecule plus instability of the tetramer in the oxy conformation. Such haemoglobin has been found in North-African populations, and the case presented here is the first one reported in Spain.

Hemoglobin Birmingham and hemoglobin Galicia: two unstable beta chain variants characterized by small deletions and insertions.

Two unstable hemoglobins (Hbs) causing rather severe hemolytic anemia have been characterized. The beta chain of Hb Birmingham, found in an adult black man, is characterized by the loss of -Leu-Ala-His-Lys- at positions 141, 142, 143, and 144 and their replacement by one Gln residue. These changes are the result of a deletion of nine nucleotides, namely two base pairs (bp) of codon 141, all of codons 142 and 143, and one bp of codon 144; the remaining CAG triplet (C from codon 141 and AG from codon 144) codes for the inserted glutamine. In the beta chain of Hb Galicia from a Spanish patient, His and Val at positions 97 and 98 are replaced by one Leu residue. This is due to an ACG deletion in codons 97 and 98, which causes the removal of one His and one Val residue, while the remaining CTG triplet (C from codon 97 and TG from codon 98) codes for the inserted leucine residue. Two mechanisms, namely slipped mispairing in the presence of short repeats, and misreading by DNA polymerase due to a local distortion of the DNA helix, are considered in explaining the origin of the small deletions.

Hb S(C)-beta+-thalassaemia: different mutations are associated with different levels of normal Hb A.

Analysis of amplified DNA through hybridization with 32P-labelled synthetic oligonucleotide probes has provided data about the molecular abnormality for beta-thalassaemic globin genes present in 32 Black and eight Mediterranean patients with Hb S(C)-beta+-thalassaemia. The patients were categorized according to these beta-thalassaemia mutations, and average haematological and haemoglobin composition data were compared for each of four different groups. Twenty-eight Black patients had the -29 A----G substitution and four had the -88 C----T substitution; all had mild disease with comparable haematology and an average Hb A level of 20%. Six Mediterranean patients had the IVS-1, 110 G----A mutation; their haematological data were nearly the same as that for the Black patients except for a lower Hb A value of 11%. Two Turkish patients with the IVS-2, 745 C----G mutation were more severely affected with mild sickling disease and low Hb A levels of 5%. Hb F levels varied greatly because of age differences; high G gamma values were observed only in patients with a beta-thalassaemia chromosome having an Xmn I site 5' to G gamma. The data readily explain the variability in Hb A level that has been repeatedly noted in patients with Hb S(C)-beta+-thalassaemia.