Cytogenetic clonality in myelodysplastic syndromes studied with fluorescence in situ hybridization: lineage, response to growth factor therapy, and clone expansion

Clonality in myelodysplastic syndromes (MDS) has been studied with various techniques including glucose-6-phosphate dehydrogenase (G6PD) isoenzyme and cytogenetic analyses, and with molecular techniques such as gene deletion studies and the analysis of restriction fragment- length polymorphisms (RFLP) of X-linked genes. In this study, we investigated the use of fluorescence in situ hybridization (FISH) with a chromosome-specific probe to examine cytogenetic clonality in peripheral blood (PB) cells from three patients with MDS. In each case, trisomy 8 was shown by conventional cytogenetic analysis at the time of the initial diagnosis. By using FISH with a probe for the centromere of chromosome 8, we identified the trisomy in individual PB cells from Wright-stained smears. With this technique, we could determine the cell lineage involved by the trisomy, and through serial analyses we could assess the response of the clonal and nonclonal cells to growth-factor therapy, and the expansion of the trisomic clone over time. In each of the three cases, various proportions of granulocytes, monocytes, eosinophils, and basophils showed trisomy 8 by FISH analysis. In none of the cases did we detect trisomy 8 in lymphocytes. By analysis of PB cells before and during therapy with recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), we found that GM-CSF stimulated both trisomic and disomic cells. During a 1-year period of sequential study, we detected an abrupt increase in the percentage of trisomic cells in one patient, a stable percentage in another, and a slowly increasing percentage in the third. The abrupt increase in the first patient preceded a transformation to a more acute phase by 2 months. We conclude that FISH analysis of PB cells of patients with MDS offers an additional approach to the study of clonality in this disorder. In some cases this analysis may provide a useful and simple means of assessing response to therapy and progression of disease.     

Development and characterization of monoclonal antiplatelet autoantibodies from autoimmune thrombocytopenic purpura-prone (NZW x BXSB)F1 mice

Male (NZW x BXSB)F1 (W/BF1) mice develop systemic autoimmunity involving autoantibodies, progressive thrombocytopenia, lupus nephritis, and degenerative coronary vascular disease with myocardial infarction. Platelet-associated IgG (PAIgG) on the platelet surface mediates platelet destruction by the reticuloendothelial system in the autoimmune thrombocytopenic purpura (ATP) of W/BF1 mice. Because the epitopes targeted in ATP by PAIgG have not been identifiable using serum from thrombocytopenic W/BF1 mice, we developed seven hybridomas secreting antiplatelet monoclonal antibodies (MoAbs) using splenocytes of thrombocytopenic W/BF1 mice. Epitopes recognized by three MoAbs were similar to those recognized by PAIgG, because eluted IgG from platelets of thrombocytopenic W/BF1 mice inhibited platelet binding by MoAbs in competitive micro-enzyme-linked immunosorbent assay. Hybridoma cells or purified Ig from the ascites of two clones (2A12 and 6A6), when injected into nude mice produced acute thrombocytopenia, elevated the levels of PAIgG, purpura, and megakaryocytosis. MoAbs of two clones also reacted with single-stranded DNA or double-stranded DNA, and one of these clones (4-13) bound to cardiolipin (CL) but was nonpathogenic in nude mice, suggesting that anti-CL and antiplatelet autoantibodies can be distinct. On immunoblotting analysis, antiplatelet MoAbs frequently bound a 100-Kd platelet protein. These MoAbs contribute to an understanding of the etiopathogenesis of ATP and the several antigens and autoantibodies involved.     

Marrow transplantation for chronic myeloid leukemia: a randomized study comparing cyclophosphamide and total body irradiation with busulfan and cyclophosphamide

A prospective randomized study was conducted comparing two conditioning regimens for the treatment of patients with chronic myeloid leukemia in chronic phase by marrow transplantation from HLA identical siblings. Sixty-nine patients received 60 mg/kg of cyclophosphamide on each of 2 successive days followed by 6 fractions of total body irradiation each of 2.0 Gy (CY-TBI), and 73 patients received 16 mg/kg of busulfan delivered over 4 days followed by 60 mg/kg CY on each of 2 successive days (BU-CY). There was no significant difference between the CY-TBI and the BU-CY groups in the 3-year probabilities of survival (0.80 for both), relapse (0.13 for both), or event-free survival (CY-TBI, 0.68; BU-CY, 0.71) or in speed of engraftment or incidence of venocclusive disease of the liver. The 4-year probabilities of survival and event- free survival for patients transplanted within 1 year of diagnosis were 0.86 and 0.72, respectively, for each group. Significantly more patients in the CY-TBI group experienced major creatinine elevations. There was significantly more acute graft-versus-host disease in the CY- TBI group. Fever days, positive blood cultures, hospitalizations, and inpatient hospital days were significantly more common in the CY-TBI group than in the BU-CY group. In conclusion, the BU-CY regimen was better tolerated than, and associated with survival and relapse probabilities that compare favorably with, the CY-TBI regimen.     

Marrow harvesting from normal donors

The experience at a single institution in harvesting marrow for allogeneic transplantation on 1,270 occasions from 1,160 normal donors is presented in detail, together with an analysis of all the donor complications. Four donors were less than 2 years old, and the youngest was 6 1/2 months. No special difficulties were encountered with these young donors. Hospitalization time was three days or less for 99% of the procedures. Six donors had life-threatening complications; three of a cardiopulmonary and two of an infectious nature, and one cerebrovascular embolic episode. Significant operative site morbidity, usually transient neuropathies, occurred in ten procedures. Ten percent of the donations were associated with transient postoperative fever of unknown origin. Increasing donor age was associated with a reduction of the cellularity of the marrow harvest. The use of stored autologous blood permitted the avoidance of blood bank transfusion in 81% of males, 69% of females, and 50% of children. It was concluded that the procedure was associated with a very low risk of complication, but that the involvement of normal donors in such an operation justifies stringent monitoring.     

Angiographic evidence that reciprocal ST-segment depression during acute myocardial infarction does not indicate remote ischemia: Analysis of 23 patients

Although reciprocal ST-segment depression from the remote noninfarcting ventricular wall during acute myocardial infarction (Ml) is a common clinical finding, the significance of this electrocardiographic pattern is unclear. Previous retrospective studies have suggested that these findings may reflect either remote wall ischemia, multivessel coronary artery disease (CAD), extensive MI or a benign electrical phenomenon. Prior studies have lacked angiographic data obtained at the time of these acute electrocardiographic changes. In this study we prospectively evaluated 23 patients with acute MI. Left ventricular wall motion, coronary anatomy and the ECG were all assessed over a short period during the acute phase of the MI. Segmental wall motion was used as a sensitive indicator of ischemia.Seventeen patients had acute anterior MI, of whom 47 % had reciprocal ST-segment depression; 6 patients had inferior MI, with 3 showing reciprocal ST depression. The mean degree of ST-segment elevation from the infarcting wall tended to be greater in patients with reciprocal ST-segment depression than in those without such reciprocal ST depression (2.8 ± 0.4 vs 1.9 ± 0.3 mm, p = 0.06). Patients with and without reciprocal ST-segment depression had similar degrees of segmental dysfunction in the infarct wall. However, no abnormalities in segmental wall motion in the remote wall were seen regardless of the presence or absence of remote wall ST-segment depression. In addition, the presence or absence of ST-segment depression did not predict the extent or degree of CAD. Finally, the magnitude of ST-segment elevation from the acutely infarcting wall correlated significantly with the degree of remote wall reciprocal ST-segment depression (r = 0.83, p < 0.01).Thus, the presence of remote wall reciprocal ST-segment depression on the ECG during the acute phase of an MI does not predict ischemia or the extent of CAD in the arteries supplying the remote noninfarcting wall. Because the reciprocal electrocardiographic changes correlate with the degree of ST-segment elevation, they probably represent a benign electrical phenomenon.     

Interobserver agreement by auscultation in the presence of a third heart sound in patients with congestive heart failure

Although the third heart sound (S3) is well recognized as an important sign in the evaluation of patients with congestive heart failure, the interobserver variability with its observation needs to be known before general applicability can be determined. Therefore, we determined the agreement among four trained observers on the presence of S3 in 81 hospitalized patients. Agreement between pairs of observers varied between 48 and 73 percent. The kappa statistic, which adjusts for agreement by chance alone, showed that agreement between various observer pairs was moderate (kappa = 0.40-0.50) at best and slight (kappa = 0.10-0.30) at worst. The rate of agreement did not appear to be affected by the time interval between measurements, by the sex of the patient or by a training effect over the time of the study. In conclusion, although S3 may be important as a clinical sign, clinicians cannot agree reliably about whether or not it is present.     

Patient-perceived barriers to antiretroviral adherence: associations with race

New antiretroviral (ARV) regimens require strict adherence if optimal suppression of HIV is to be maintained. This study is a theory-based examination of racial differences in patient-perceived barriers and reported ARV adherence. Participants (N=149) completed the Patient Medication Adherence Questionnaire (PMAQ), measuring adherence and perceived barriers to adherence. Adherence was defined as a self-report of 100% adherence in the past four weeks. Odds ratios were calculated to determine the relation of reported barriers to adherence for race and gender groups, and for the sample overall. For every ten-point increase in barrier score, there was an 86% increased risk of being non-adherent (OR=1.86; 95% CI: 1.19, 2.91). Adherence was not different between racial and gender groups, nor was total barrier score. However, individual barriers were differentially endorsed across groups. Rather than relying on demographic predictors, which may be only an indirect marker of adherence, evaluations of adherence should examine the psychological and social barriers to positive adherence outcomes in individual patients. Our findings support the use of theory-based behavioural interventions that address perceived barriers to adherence and other health promotion activities.