Evidence for immunoglobulin heavy chain variable region gene replacement in a patient with B cell chronic lymphocytic leukemia.

Immunoglobulin heavy chain variable (V) gene replacement is an unusual recombinatorial event characterized by rearrangement of a germline V gene to a preformed VDJ gene complex. This phenomenon has occasionally been implicated in the emergence of clonal subpopulations during the course of acute lymphoblastic leukemia; it has also been found in murine precursor B cell lines. V gene replacement has never been described in lymphoproliferative disorders corresponding to more differentiated stages of B cell ontogeny. The present communication provides evidence for the operation of the same mechanism in B cell chronic lymphocytic leukemia (B-CLL). Genomic DNA and total cellular RNA extracted from peripheral blood mononuclear cells of a 48-year-old female patient diagnosed as having typical B-CLL were subjected to polymerase chain reaction (PCR) amplification aiming to detect rearranged clonal heavy and light chain variable genes (VH and VL, respectively). PCR consistently gave two VH amplification products, both at the DNA and the RNA level; similar analysis for the VL region revealed the presence of a single rearranged VK gene. Direct sequence analysis of the PCR products revealed that, except for a number of silent mutations, the single rearranged VK gene was identical to the germline A1-A17 VK gene. The two rearranged VH gene segments belong to the VHl and VHIII gene families and are closely homologous, respectively, to the germline gene segments V1-18 and V3-30, which have been shown to be used by autoantibodies. Both rearranged VH genes showed identical in-frame D-N-JH junctions and JH gene usage (JH5b), whereas the VH-N-D junctions were different. The above findings indicate that, during the course of the disease of our patient, VH gene replacement took place giving rise to two different clonally related subpopulations. This raises the intriguing possibility that the recombinase machinery, which governs Ig recombinatorial processes, might be operative even at more advanced stages in B cell ontogeny.

Pharmacological induction of fetal hemoglobin in sickle cell disease and beta-thalassemia.

A number of pharmacological agents are currently available for the induction of fetal hemoglobin (HbF) in patients with sickle cell disease and beta-thalassemia. Here we review the development of this new class of therapeutics and summarize the clinical trials that investigate their efficacy in patients with hemoglobin disorders. Hydroxyurea is the first of these drugs to be approved by the Food and Drug Administration for the treatment of sickle cell disease. Currently, the major focus is the development of safer agents and combinations of drugs that can increase HbF to levels high enough to prevent all complications of the disease. Progress in adapting the same strategy to the treatment of thalassemic disorders has been much slower. Although all the agents that are effective in sickle cell disease have similar HbF-inducing activity in beta-thalassemia, their use has rarely resulted in significant amelioration of the anemia. More research and more effective agents will be needed to make a significant impact on thalassemia. Nonetheless, success in this relatively young field has been very gratifying; before the end of this decade, clinically meaningful induction of HbF may become an achievable goal in most patients with hemoglobin disorders.

Structural study of hemoglobin Knossos, beta 27 (B9) Ala leads to Ser. A new abnormal hemoglobin present as a silent beta-thalassemia.

A new electrophoretically silent hemoglobin variant is described that produces the classical phenotype of beta thalassemic intermedia in association with beta thalassemia trait. This variant has the expression of a silent beta thalassemia trait. The abnormal hemoglobin was detected by acid-urea-Triton-acrylamide electrophoresis and further demonstrated by isoelectric focusing. The amount of the variant in carrier is approximately 30% of the total hemoglobin. No instability was found. Absence of hemoglobin A in the propositus blood facilitated structural studies. Peptides maps were normal but analysis of individual peptide spots showed an Ala leads to Ser substitution in the beta T3. This variant has been previously called Hb Knossos (beta 27 (B9) Ala leads to Ser).

Exercise-induced myocardial perfusion abnormalities in sickle beta-thalassemia: Tc-99m tetrofosmin gated SPECT imaging study.

PURPOSE: To determine the mechanism of myocardial ischemia in patients with sickle beta-thalassemia, we performed a scintigraphic evaluation of myocardial perfusion during exercise. SUBJECTS AND METHODS: We studied 30 patients with sickle beta-thalassemia, (mean [+/-SD] age, 37 +/- 10 years) who had no electrocardiographic (ECG), radiographic, or echo-Doppler signs of pulmonary hypertension, left ventricular hypertrophy, or impaired contractility. All patients had a hemoglobin level greater than 7 g/dL. Treadmill exercise test was performed according to the Bruce protocol. Myocardial perfusion was assessed by single-photon emission computed tomography, using Tetrofosmin Tc-99 m Myoview as radiotracer, at peak exercise and again 4 hours later. RESULTS: Eight patients (27%) developed stress-induced scintigraphic perfusion abnormalities that were reversible in all but 1 patient. Subsequent coronary angiograms were normal in all 8 patients. ST segment depression was seen during exercise in 5 of the 7 patients who had reversible perfusion defects. Except for a significantly greater white blood cell count, these 5 patients did not differ from the rest of patients by sex, age, hemoglobin level, percentage hemoglobin F, beta-thalassemia genotype, or risk factors for coronary artery disease. Three of the 5 patients with perfusion and ECG abnormalities (and another with only perfusion defects) developed a stress-induced sickling crisis. CONCLUSION: Physical stress may induce myocardial ischemia in sickle beta-thalassemia patients with normal coronary arteries and elicit painful crises. The sickling process, activated by exercise, could be the common underlying mechanism.

Pure red cell aplasia associated with true thymic hyperplasia.

Pure red cell aplasia was diagnosed in a 35-year-old otherwise healthy woman. Chest computed tomography (CT)-imaging investigation, detected an upper mediastinal mass corresponding to the thymus gland. A thoracotomy was performed and an enlarged thymus mass was removed, rapidly followed by a full hematologic recovery. Thymic histology confirmed a significant degree of hyperplasia. We conclude that not only thymomas but also other types of thymic pathology may be associated with this type of hematologic dyscrasia.

Pulmonary hypertension and right heart failure in patients with beta-thalassemia intermedia.

We analyzed seven patients with beta-thalassemia intermedia presenting with congestive heart failure secondary to pulmonary hypertension. This condition has been recognized only recently as part of the clinical spectrum of beta-thalassemia. Our group of patients included two men and five women with the clinical picture and laboratory data typical of beta-thalassemia intermedia. The mean age was 37.7 +/- 11.4 years, mean hematocrit value was 28.5 +/- 1.8%, mean number of transfused blood units was 171 +/- 153, and mean serum ferritin levels were 4,428 +/- 2,006 ng/mL. All but one of these patients had undergone splenectomy. Common findings of the investigative procedures include the following: dilation of the main pulmonary artery and cardiac enlargement in the chest radiograph; signs of right ventricular hypertrophy in the ECG; and dilated right ventricle with good left ventricular function in the echo study. Right heart catheterization showed the pulmonary systolic pressure to range from 55 to 90 mm Hg (74.1 +/- 10.3), pulmonary diastolic pressure from 25 to 50 mm Hg (37.7 +/- 8.7), mean pressure from 35 to 60 mm Hg (49.7 +/- 7.9), and pulmonary vascular resistance from 267 to 667 dynes.s.cm-5. Pulmonary capillary wedge pressure was within the normal range of values. The pathophysiologic condition of pulmonary hypertension in these patients is most probably associated with beta-thalassemia. There are mechanisms that increase cardiac output and at the same time restrict the pulmonary vascular bed. The results of this study imply that treatment decisions should be reconsidered for such patients.

Increased erythropoietin level induced by hydroxyurea treatment of sickle cell patients.

INTRODUCTION: Administration of hydroxyurea in sickle cell disease is associated with a dramatic increase of HbF along with a significant clinical improvement and, occasionally, increased total hemoglobin levels. The underlying mechanisms are not yet fully elucidated. MATERIALS AND METHODS: We report the response of three patients with homozygous sickle cell disease and 10 patients with compound HbS/beta-thalassemia (four with beta(o)thal/HbS and six with beta(+)thal/HbS respectively) to hydroxyurea treatment with regards to their serum erythropoietin levels (sEpo). RESULTS: Baseline sEpo levels varied from 33.0 to 284.0 IU/L and showed a significant negative correlation with the respective Hb values (P<0.007). Two to three weeks after initiation of treatment, the sEpo values started to increase and reached levels three to 31 times higher than the baseline two to three weeks later. Thereafter, in most cases the Epo values decreased and remained at intermediate levels throughout the rest of hydroxyurea administration, while in a few cases, they returned to baseline. An inappropriate increase of sEpo following treatment with various cytostatic drugs, independently of anemia induced by cytostatic agents, has already been reported in the literature. The cytostatics included cyclophosphamide, anthracyclines, cytosine arabinoside etc., but not hydroxyurea. The results described here with hydroxyurea are virtually similar, ie, they show a significant sEpo increase five to ten days post therapy with no apparent cause. Pulses of high dose Epo have been reported to promote proliferation of erythroid precursors with HbF synthesizing capacity. CONCLUSION: Our hypothesis is that a similar phenomenon may occur here also, in the sense that peaks of endogenous Epo may promote proliferation of erythroid precursors which maintain the capacity to synthesize HbF.

Detection of CD55- and/or CD59-deficient red cell populations in patients with lymphoproliferative syndromes.

INTRODUCTION: Paroxysmal nocturnal hemoglobinuria is an acquired clonal stem cell disorder characterized by the decrease or absence of glycosylphosphatidylinositol-anchored molecules from the surface of the affected cells, such as CD55 and CD59, resulting in chronic intravascular hemolysis, cytopenia and increased tendency to thrombosis. PNH-phenotype has been described in various hematological disorders, mainly in aplastic anemia and myelodysplastic syndromes, while it has been reported that complete deficiency of CD55 and CD59 has also been found in patients with lymphoproliferative syndromes, like non-Hodgkin's lymphomas. MATERIALS AND METHODS: The presence of CD55- and/or CD59-defective red cell populations was evaluated in 217 patients with lymphoproliferative syndromes. The study population included 87 patients with NHL, 55 with HD, 49 with CLL, 22 with ALL and four with hairy cell leukemia. One hundred and twenty-one healthy blood donors and seven patients with PNH were also studied as control groups. The sephacryl gel microtyping system was performed for the detection of CD55- and CD59-deficient red cell populations. Ham and sucrose lysis tests were also performed in all samples with CD55 or CD59 negative populations. RESULTS: Red cell populations deficient in both CD55 and CD59 molecules were detected in 9.2% of patients with lymphoproliferative syndromes (more often in ALL and nodular sclerosis type of HD) and in all PNH patients. CD55-deficient red cell populations were found in 8.7% of LPS patients (especially in low grade B-cell NHL), while CD59-deficient populations were found in only two patients with low grade B-cell NHL. CONCLUSION: These data indicate a possible association between paroxysmal nocturnal hemoglobinuria phenotype and lymphoproliferative syndromes, while further investigation is necessary to work out the mechanisms and the significance of the existence of this phenotype in these patients.

Proteolysis in thalassemia: studies with protease inhibitors.

Intracellular proteolysis was studied in both thalassemic and normal reticulocytes. The main experiment comprised a short incubation of RBC with [3H] leucine, extensive washings, and further incubation in the presence of cold leucine and protein synthesis inhibitors. In the aliquots removed at various time intervals, the TCA-soluble radioactivity increased in contrast with the TCA-precipitable cpm, which decreased with no exception on prolonged incubation. Results were more pronounced when the initial incubation was carried out in the presence of a valine analog and were markedly inhibited when phenanthroline was added during the second phase of the experiment. Cell-free system gave similar results. "globin" containing analog residues were degraded easier than free alpha- and beta-globin chains; free alpha or beta Hb chains are not proteolysed. Thalassemic and normal reticulocytes did not show any significant differences. It is proposed that the thalassemic RBC contain efficient proteolytic mechanisms up to the end of their maturation, but these prove incapable of proteolysing the excess alpha chains completely. Chemical manipulations aiming to make those chains more digestible constitute another approach to the treatment of thalassemia.

Hydroxyurea therapy in thalassemia.

The clinical effectiveness of Hydroxyurea in thalassemia is still controversial. The present paper puts together the authors' experience in two groups of patients with thalassemia intermedia and sickle cell/beta-thalassemia treated with varying dosages of hydroxyurea over several months. A third group received hydroxyurea along with recombinant human erythropoietin. Our observations are summarized in that treatment with hydroxyrea results in a significant increase of fetal hemoglobin with no change of the total hemoglobin levels. The drug causes also a considerable increase of the erythrocyte volume and hemoglobin content while the MCHC values remain unchanged. As a rule, and without objective criteria so far, patients state feeling better and having more energy. The authors postulate that this feeling may reflect the significant decrease of ineffective erythropoiesis resulting by the replacement of the poorly hemoglobinized, prematurely dying erythroid progenitor and red cell population by another population of cells with higher hemoglobin content and longer survival, the regeneration of which requires less energy and consumption. As expected, patients with sickle cell/beta-thalassemia have also fewer crises and painful episodes. The above findings are in keeping with the few available reports in the literature.

A C----G mutation at nt position 6 3' to the terminating codon may be the cause of a silent beta-thalassemia.

We describe the hematological and clinical data for a young Greek patient with beta-thalassemia intermedia and for several members of her family. The patient had inherited the common IVS-I-1 (G----A) mutation from her mother, while the second beta-globin gene had a C----G mutation at position 6 3' to the terminating codon (term. + 6). Her father and three additional relatives with a heterozygosity for this newly discovered mutation had no hematological abnormalities, normal Hb A2 values, and a nearly normal in vitro chain synthesis ratio. Analyses of nearly 500 additional beta-thalassemia and normal chromosomes failed to detect this mutation which eliminates it as a common polymorphism. Although our findings may indicate a rare polymorphism, the probability that it represents the cause of diminished beta chain synthesis is very high indeed. We suggest that the C----G mutation in this untranslated region of the beta-globin gene causes a slight decrease in the stability of the mRNA which becomes clinically important only in situations when beta chain synthesis in trans is eliminated.

Evaluation at single cell level of residual Philadelphia negative hemopoietic stem cells in chronic phase CML patients.

In chronic myeloid leukemia, accurate determination of Ph(-) Hemopoietic stem cells (HSC) in peripheral blood (PB), bone marrow (BM) and leukapheresis products is important for the selection of patients for whom mobilization, collection, and autografting of Ph(-) HSC are envisaged. To this effect, the BCR/ABL fusion was assessed at the single cell level in 25 sets of PB and BM samples using dual-color I-FISH in immunophenotyped CD34(+) cells and RT-PCR of individual CFU-GM colonies. In 15 cases found to be 100% Ph(+), the respective BCR/ABL gene was absent in 30% of CD34(+) cells, while the respective transcripts could not be identified in 17% of CFU-GM. The mean percentage of BCR/ABL(-) CD34(+) cells and CFU-GM cells was higher (38% and 29%, respectively) in untreated patients than in treated patients (24% and 7%, respectively). In eight cases with cytogenetic response (CgR), the percentage of Ph(-) metaphases correlated with the level of BCR/ABL(-) colonies in BM and PB and with the proportion of BCR/ABL(-) CD34(+) cells in the BM. Immunophenotyping and FISH was fast, easy, always informative, and quantitative for the BCR/ABL(-) CD34(+) cells. Our results show that (a) at early diagnosis a high frequency of BCR/ABL(-) HSC circulate in the PB and that Ph(-) hematopoiesis is not completely suppressed; (b) although normal clonogenic cells decline rapidly within a few months after diagnosis, appreciable numbers of normal CD34(+) cells survive in chronic phase, especially in patients with CgR.

Hypereosinophilia associated with monosomy 7.

Cytogenetic analysis of bone marrow cells from a patient with anemia, marked leukocytosis with eosinophilia, and thrombocytopenia showed monosomy 7 in all metaphases examined. The patient has refractory anemia (RA) according to FAB classification. Because of the hypereosinophilia of the patient, PCR technique was performed and no bcr-abl mRNA, specific for chronic myelogenous leukemia, was detected. Monosomy 7 has not been previously described in cases with hypereosinophilia. We assume, according to previous reports, that multiple genetic lesions can be involved in the pathogenesis of hypereosinophilia in this patient.

A novel chromosomal abnormality involving chromosomes 2 and 18 in a patient with myelodysplastic syndrome.

Cytogenetic analysis of bone marrow cells from a patient with myelodysplastic syndrome associated with eosinophilia showed a complex translocation with a 46,XY,t(2;18;2)(p23;q11;q32) karyotype. The patient has refractory anemia (RA) according to the French-American-British Cooperative Group (FAB) classification, and after 90 months of follow-up he shows no evidence of leukemic transformation. This chromosomal abnormality has not been previously described in myelodysplastic syndromes and may be associated with good prognosis as the patient has been stable for a long time.

Molecular demonstration of BCR/ABL fusion in two cases with chronic myeloproliferative disorder carrying variant Philadelphia t(14;22)(q32;q11).

We report two cases with chronic myeloproliferative disorder which were found to carry simple variant Philadelphia (Ph) t(14;22)(q32;q11) in unstimulated bone marrow mononuclear cells. Both cases were characterized molecularly by Southern blot, reverse transcription-polymerase chain reaction (RT-PCR), and direct sequencing of the RT-PCR products. In the first case (female, aged 65, in blastic transformation which developed one year after the initial diagnosis of myelofibrosis), a t(14;22) (q32;q11) was found in association with several other chromosomal abnormalities [48,XX,+X,+5,del(5) (q12q32),+8,der(9)t(9;11)(q32;q11),-11]; molecular analysis demonstrated the presence of a BCR-ABL chimeric gene and mRNA transcript of the b2-a2 type. In the second case (female, aged 16, with clinical and hematologic features typical of chronic myelogenous leukemia in chronic phase), a t(14;22) (q32;q11) was identified as the sole karyotypic abnormality; again, molecular analysis demonstrated the presence of a BCR-ABL chimeric gene and mRNA transcript, this time of the b3-a2 type. Our findings further support the notion that, even when undetectable by conventional cytogenetics, band 9q34 participates in all Ph chromosomes and leads to the formation of chimeric BCR-ABL genes.

Trisomy 11 with loss of the Y chromosome and trisomy 13 in a case of de novo acute myeloid leukemia.

We report a man with de novo acute myeloid leukemia (M4 of the FAB classification) bearing two abnormal clones in the bone marrow cells. The clones showed trisomy 11 with loss of the Y chromosome and trisomy 13, respectively.

Correlation of the site of M-bcr breakpoint with chronic phase duration, blastic crisis lineage and thrombocytosis in Ph1-positive chronic myelogenous leukemia.

The breakpoints on chromosome 22 in CML occur within a 5.8 kb region called the Major breakpoint cluster region (M-bcr). DNA mapping within the M-bcr region was performed in 41 long term followed Ph1-positive CML patients using the Southern blot technique. The purpose of this study was to determine whether localization of M-bcr breakpoint correlates with the length of chronic phase of the disease, blastic crisis lineage and thrombocytosis at the time of initial diagnosis. Our results fail to indicate any correlation between breakpoint localization and duration of chronic phase, blastic crisis lineage and platelet count at diagnosis.

Detection of "PNH red cell" populations in hematological disorders using the Sephacryl Gel Test micro typing system.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder characterised by an unusual sensitivity of abnormal red cell population(s) to complement lysis, due to a complete or incomplete defect of various surface molecules, including CD55 and CD59. PNH has been associated with various hematological disorders. Using a newly introduced method, the Sephacryl gel test microtyping system, we investigated the presence of CD55 or CD59 defective red cell populations in several hematological disorders. It was also found that a large proportion of such patients possess CD55 deficient populations, while a smaller but still significant proportion possess CD59 deficient populations. Defective red cell populations were detected in normal subjects as well. These findings need further investigation. Nevertheless the Sephacryl Gel Test microtyping system although non specific, seems to be useful in screening for the PNH and/or "PNH-like" red cell defect in several hematological disorders.

Angioid streaks in sickle-thalassemia.

Angioid streaks have been described in a diverse group of diseases including hemoglobinopathies such as sickle cell anemia and beta-thalassemia. We investigated the prevalence of angioid streaks and pseudoxanthoma elasticum in the rare situation of patients who had compound heterozygous traits for hemoglobin S and beta-thalassemia. We examined 58 consecutive patients with sickle-thalassemia. Of these, 25 were men and 33 were women, and they ranged in age from 19 to 58 years (mean, 32.6 years). Angioid streaks were identified in six of 58 patients (10%), and of these three also displayed the cutaneous lesions of pseudoxanthoma elasticum, which were confirmed by skin biopsy. An expanded study on several relatives of the patients with angioid streaks failed to identify any similar cases. Statistical evaluation of the main hematologic and biochemical parameters in the patients with and without angioid streaks did not demonstrate any significant differences, except that the thalassemic component in all six patients with angioid streaks was beta(0) (that is, did not allow the synthesis of hemoglobin A). We conclude that angioid streaks and pseudoxanthoma elasticum skin lesions occur with an increased frequency in patients with sickle-thalassemia.

Doppler-determined peak systolic tricuspid pressure gradient in persons with normal pulmonary function and tricuspid regurgitation.

The Doppler-estimated peak systolic tricuspid pressure gradient is the most reliable noninvasive method for the evaluation of pulmonary artery systolic pressure in patients with tricuspid regurgitation. Our goal was to evaluate the range of this gradient in healthy persons and determine a normal upper limit. We studied 53 healthy persons (34 women, 19 men; aged 14 to 55 years, mean 38.9 +/- 12.7 years) who did not smoke and who had an adequate Doppler signal of tricuspid regurgitation. The presence of pulmonary or cardiac disorders was excluded by a review of the subject's medical history in addition to physical examination, spirometry, arterial blood gasses determination, electrocardiography, chest x-ray examination, and rest echocardiography. Tricuspid gradient ranged from 12.6 to 29. 3 mm Hg (mean 19.3 +/- 4.0); 35.8% of patients had values higher than 20 mm Hg. In conclusion, a tricuspid gradient of 30 mm Hg may be considered as the upper normal limit. The different approaches for estimating mean right atrial pressure are also discussed.