Biomedical subjects
Publications and source records attributed to T Ruutu.
We describe fatal rhabdomyolysis in a 24-year-old man who had received a bone marrow transplant from a sibling for the treatment of chronic granulocytic leukaemia. The rhabdomyolysis was preceded by a grand mal seizure probably caused by a combination of cyclosporin A and corticosteroids given for the prevention and treatment of acute graft-versus-host disease.
Cytogenetic studies detected abnormalities in 107 (43%) of the 247 patients in this series. Some degree of overt clinical progression occurred in 55 patients (22%), this being 29% of those patients with cytogenetic abnormalities and 17% of those with normal chromosomes. The presence and complexity of a clonal cytogenetic abnormality correlated with shorter survival. In each clone category of a complexity classification (simple, complex, very complex), patients with some normal cells appeared to have better survival than those with none. In multiple regression analyses, the prognostic value of chromosomes was independent of (and second in importance to) the FAB type of myelodysplastic syndrome (MDS) whichever chromosome classification was used. Patients with refractory anemia (RA) had the lowest incidence of chromosome abnormalities and no cases were found to have only abnormal cells (AA). A greater proportion of patients with refractory anemia with an excess of blasts (RAEB) and RAEB in transformation (RAEB-t) had clonal abnormalities. Morphology alone is not at present able to distinguish between RA or refractory anemia with ringed sideroblasts and similar disorders that may not be MDS in the strict sense. Demonstration of a clonal cytogenetic abnormality remains a positive indication of the presence of the neoplastic nature of the disease.
To evaluate the importance of pretreatment karyotype in predicting long-term outcome in acute lymphoblastic leukemia (ALL), we performed a follow-up study of the 329 patients from the Third International Workshop on Chromosomes in Leukemia. Living patients have now been followed a minimum of 6 years. Patients were divided into ten groups according to pretreatment karyotype: no abnormalities, one of the following structural abnormalities [the Philadelphia chromosome, rearrangements involving 8q24, t(4;11), 14q+, 6q-] or, in the remaining cases, modal number (less than 46, 46, 47-50, greater than 50). As previously reported for achievement and duration of complete remission, and overall survival, disease-free survival differed significantly (p less than 0.001) among chromosome groups for both adults and children. Among children, karyotype was an independent prognostic factor for predicting disease-free survival. Because of the long follow-up, we now have been able to utilize statistical models to estimate the percentage of patients cured, according to karyotype alone and combined with other risk factors. Adults with the highest likelihood of cure (21-33%) were those patients with FAB-L1, a leukocyte count of 50,000/microliters or less, and one of the following chromosome groups: greater than 50, 47-50, 6q-, or normal. In children these same characteristics were associated with the highest percentage of cure (58-71% cured). In addition, we identified several groups of children with less than 15% chance of cure who clearly need to be treated as high-risk patients at diagnosis. Future studies of patients who have received risk-adapted therapy based on these chromosome data are needed to determine if more intensive treatment will improve the outlook of patients with cytogenetically unfavorable types of ALL.
The two methods commonly used to diagnose cytomegalovirus (CMV) infections, conventional tissue culture and detection of early CMV nuclear antigen by immunofluorescence from cell culture, were performed in parallel on 597 clinical specimens. CMV was detected by the early-antigen test in 108 samples, of which 102 (94%) were detected 1 to 3 days after inoculation. Of these 108 CMV-positive specimens, seven were negative on conventional culture. Two samples negative in the early-antigen test were positive on conventional culture. Thus, CMV was detected in 110 specimens. A cytopathic effect in conventional tissue culture occurred 9 to 42 days after inoculation. The diagnosis of CMV infection was possible by the conventional method 29.6 +/- 12.7 days and by early-antigen immunofluorescence 1.9 +/- 1.5 days after obtaining the specimen. The rapid early-antigen test was slightly more sensitive than culture, and fewer samples were lost due to bacterial or fungal infections during incubation. Detection of CMV by conventional culture usually requires several weeks and provides a diagnosis only retrospectively. The main advantage of the early-antigen test is that a virologically proven diagnosis of CMV infection is available at an early stage.
DNA-fingerprint (DNA-F) analysis, based on the polymorphism of the tandem repeats of minisatellite areas in human genome, has a capacity to reveal minor changes in dispersed areas of human genome. In this study we have applied DNA-F analysis to the detection of differences between leukemic phase and remission in acute myeloid leukemia (AML). In order to identify normal and leukemic cell populations we used two molecular probes: Jeffreys' minisatellite probes 33.6 + 33.15 and M13 wild-type phage probe. Comparison of varying minisatellite fragments between remission and diagnosis/relapse was performed by Southern blot hybridization in 21 patients with AML. The results demonstrate that Southern hybridization with minisatellite probes can detect differences in DNA-fingerprints between leukemic phase and remission in 44% of AML patients. Thus differences in DNA-fingerprinting provides a new molecular marker, which can be useful in the detection of residual disease as well as in the study of the pathogenesis of AML.
The effect of haem arginate, a new stable haem compound, was studied on colony formation by erythroid (CFU-E, BFU-E) and granulocyte-macrophage (CFU-GM) progenitors from the bone marrow of 12 healthy persons. At the concentration of 10 mumol/l haem arginate significantly (p less than 0.05) increased and at the concentration of 200 mumol/l significantly decreased (p less than 0.005) the colony formation of CFU-E. A similar, though not significant, trend was seen in the BFU-E colony growth, while the CFU-GM colony formation was not influenced at any concentration between 1 and 200 mumol/l. We conclude that the stimulation of erythropoiesis in vitro is seen at lower concentrations with haem arginate than with former haemin preparations.
Acute graft-versus-host disease (aGVHD) of the liver was studied with fine-needle aspiration biopsies of thirty-four bone marrow transplant recipients. White cell differentials of liver FNABs and simultaneously taken blood samples were performed, and the increment and corrected increment methods were used to quantitate the inflammatory reaction in the liver. Biopsies taken before transplantation were used as the baseline. During aGVHD, the percentage of lymphoid cells and monocytes increased in the liver. The appearance of immunological blasts, together with a high proportion of activated lymphocytes in the FNABs, were typical findings during aGVHD. In patients with apparent prolonged liver graft-versus-host disease small lymphocytes were the predominating cell type. After initiating corticosteroid treatment, the number of blasts and the proportion of activated lymphocytes decreased. There was no significant difference in the proportions of CD4- and CD8-positive lymphoid cells in FNABs during or after aGVHD.
Three patients with T-cell lymphoma were studied with a combined cytogenetic-immunological analysis (MAC) as well as by Southern blotting analysis. The MAC method, which allows simultaneous study of both chromosomes and immunophenotype of a mitotic cell, revealed that in each case the karyotypically abnormal and, therefore, neoplastic cells originated from different stages of T-cell maturation. In one case the abnormality was seen in cells expressing CD3 and CD10, in the second in cells expressing CD3 and CD8, and in the third case in cells expressing CD4 but not CD3. Mitoses were frequent also in non-neoplastic cells, but these mitoses were normal. Southern blotting analysis revealed TCR beta rearrangements in all patients. In addition, one patient displayed TCR gamma rearrangement, and one patient a rearrangement in the IgH joining region.
Erythroid colony formation in vitro was studied in 80 patients with erythrocytosis. 43 of the patients had polycythaemia vera (PV), 18 patients had secondary erythrocytosis, 6 had normal red cell mass, and 13 patients were regarded as unclassified. Spontaneous erythroid colony formation, in the absence of exogenous erythropoietin in the cultures, was discovered in all patients with PV, whereas no patient with secondary erythrocytosis or with normal red cell mass showed this phenomenon. 8 of the 13 patients with unclassified erythrocytosis spontaneously formed erythroid colonies. 7 patient with unclassified erythrocytosis have been followed for 5 yr. 3 of the 4 patients with spontaneous colony growth but none of the 3 without it can now be classified as PV. Thus, spontaneous erythroid colony formation indicates PV even in early and atypical cases. Therefore, the culture of erythroid progenitors is very useful in the differential diagnosis of problematic cases with erythrocytosis.
In vitro megakaryocytic colony formation by progenitors from the bone marrow was studied in 40 myelodysplastic syndrome patients. Megakaryocytic colonies were decreased in number or absent in 30 patients; only 10 showed normal colony growth. The growth correlated to some extent with the FAB-class. Patients with normal colony formation had either RA or RARS, but also in these two FAB-types half of the patients showed reduced megakaryocytic colony formation. Only 1 out of 15 patients with RAEB or RAEBt had normal megakaryocyte growth. The patients with CMML did not show any megakaryocytic colonies. The growth of erythroid colonies was normal in 3 patients and reduced or absent in the others. All 3 with normal erythroid colony formation also showed normal megakaryocyte growth, and all patients with normal megakaryocyte colony formation also had normal granulocyte-macrophage colony growth. Granulocyte-macrophage colony and cluster formation was normal in 17 patients. Defective formation of megakaryocytic, erythroid, and granulocyte-macrophage colonies was seen in 22 patients, compatible with a defect in a pluripotent stem cell. Megakaryocytic colony formation had no obvious correlation with any specific chromosome abnormality, and the distribution of the growth patterns was almost similar in patients with a normal and those with an abnormal karyotype.
Deletions in the long arm of chromosome 7 are common recurrent abnormalities in secondary leukemias and myelodysplastic syndromes. To learn more about the basic mechanisms involved, we used Southern blot analysis to study four patients with different 7q--deletions to determine the exact break-points and to define the extent of the deletions. Several genes and DNA sequences from 16 different loci were found to be deleted, as judged by the absence or considerable weakening of an allelic band in granulocytic DNA in patients with constitutional heterozygosity. A terminal segment was present in each of the partially deleted chromosomes, as shown by heterozygosity for probes from the region 7q35----qter in granulocyte DNA. This indicated that the chromosome 7q deletions were interstitial, rather than terminal, in each of these patients. The length of the preserved terminal segment varied among the patients. Our results support gene loss as a mechanism contributing to leukemogenesis. Since the deletions are interstitial, hybrid genes may be formed at the junction, but the variation in breakpoints argues against the existence of a common hybrid gene of importance to the malignant process.
The involvement of the erythropoietin (EPO), plasminogen activator inhibitor type I (PAI1), and multi-drug resistance (MDR2) genes located in chromosomal region 7q21-22 was studied in patients with myeloid disorders and with or without a chromosome 7 abnormality. Separated blood mononuclear cells and granulocytes from 21 patients were used in restriction fragment length polymorphism (RFLP) studies with gene-specific DNA probes. A marked weakness of one of the allelic bands was observed in granulocyte-derived DNA from heterozygous patients with monosomy 7. In four patients with a partial deletion of chromosome 7 long arm (7q-), marked weakness of an allelic band was observed in granulocyte-derived DNA with PAI1 probe (four heterozygous patients) and MDR2 probe (one heterozygous patient), implying deletion of these genes. In contrast, the EPO gene was not deleted in these patients, as demonstrated by the presence of two allelic bands of equal strength in granulocyte-derived DNA (two patients) or by gene dosage estimation (two patients). Two allelic bands of equal strength were also observed in three heterozygous patients with an arbitrary probe (pKV13) located in 7cen-q21.3. Unexpected hemizygosity or hybridization bands were not observed in any patient. We conclude that PAI1 and MDR2 are located distally of EPO in 7q22, and that none of these genes is commonly rearranged in myeloid disorders. The chromosome 7 long arm deletion breakpoint is located in a relatively narrow segment between the PAI1 and EPO genes in different patients. The deletion may involve a specific site in DNA, since the genetic distance between the PAI1 and EPO genes is only 3 cM.
A non-radioactive in situ hybridization method was used to detect residual host cells after bone marrow transplantation (BMT). A biotinylated Y-specific DNA probe was hybridized to bone marrow cells of three male patients who had received a bone marrow graft from their sisters for the treatment of acute leukaemia. Host cells were detected in two of the three patients, both in clinical remission, in six and seven cells out of 1000 cells analysed, respectively. Only one of a total of 3000 cells from three female controls showed a 'Y-signal'; 2580 cells out of a total of 3000 cells from three male controls showed a positive signal. These results indicate that in situ hybridization is a reliable and sensitive method for the detection of host cells after BMT.
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We have stored at -196 degrees C peripheral blood buffy coat (BC) and bone marrow (BM) cells collected from 47 patients with chronic granulocytic leukaemia in the chronic phase. Dimethyl sulphoxide (DMSO) 10% was used as cryoprotective agent. As these cells include CFUc and probably pluripotential stem cells they may be transfused as part of the management of patients who enter blast cell transformation. The mean numbers of nucleated cells collected and stored per procedure was about 9 times greater for BC collections than for BM harvests (106 +/- 49 (SD) X 10(9) versus 11.9 +/- 6.6 X 10(9) respectively). Agar CFUc assay showed that stored cells may remain viable for up to 5 years. Since in vitro studies showed that CFUc proliferation is not inhibited by low concentrations of DMSO the removal of all DMSO during cell reconstitution before transfusion may not be necessary. If autologous BC cells are capable of repopulating the BM of patients treated for CGL in blast cell transformation the routine collection and storage of BC rather than BM cells may be desirable for all newly diagnosed patients.