Variable growth in vitro of chromosomally different stem cells in myelodysplastic syndromes.

Ten patients with myelodysplastic syndromes were investigated using in vitro colony assay of bone marrow cells and chromosome analysis of single colonies. The result was compared with conventional cytogenetic analysis of bone marrow cells. The chromosome abnormalities included were 5q-, +8, -7, 11q-, -Y and one complex karyotype. Erythroid colony formation was reduced in eight patients, while the number of granulocyte-macrophage colony-forming units was normal or increased. Cytogenetic examination of single colonies showed that both chromosomally normal and abnormal stem cells had colony forming ability. The proportion of cytogenetically abnormal colonies varied between 25 and 100% of analysed colonies. No further clones than those found in direct cytogenetic analysis were revealed after in vitro growth. One patient with a 5q- abnormality and one patient with a -7 abnormality showed a significantly lower proportion of cytogenetically abnormal colonies than the proportion of abnormal cells in the direct bone marrow chromosome preparation. One patient with a +8 abnormality showed a growth advantage of the +8 clone in comparison with the chromosomally normal clones, but this was not statistically significant. In two patients a chromosomally changed stem cell gave rise to both erythroid and myeloid colonies. The FAB-class did not seem to influence the growth of either chromosomally normal or abnormal colonies.

Masked monosomy 7 in myelodysplastic syndromes is uncommon and of undetermined clinical significance.

Masked monosomy 7, i.e. detected by FISH but not by conventional cytogenetics, has been reported in varying frequency in MDS. To establish the prevalence and possible clinical significance of the aberration, we studied the 123 previously karyotyped MDS patients using FISH and a DNA probe specific for chromosome 7. Metaphase cytogenetics revealed ten patients (8%) with monosomy 7 (6 RAEB and 4 RAEB-t). FISH confirmed this result and detected four more cases (4%) with masked monosomy 7 (3 RA and 1 RARS). Thus, masked monosomy 7 is less common than has been suggested, and does not seem to carry the same prognostic weight as monosomy 7 diagnosed by metaphase cytogenetics.

Dysplastic megakaryopoiesis with thrombocytopenia and chromosomal aberration.

A case of isolated thrombocytopenia with decreased platelet production and abnormal megakaryopoiesis is described. In the bone marrow, a chromosomally aberrant clone, 45,XX,-11,-18,+der (11;18)(11q13;18p11), was found. These findings indicate a myelodysplastic nature of the abnormal thrombocytopoiesis. The described case demonstrates the value of a bone marrow examination including histopathology with immunologic techniques to evaluate the megakaryopoiesis in thrombocytopenia and the interest of cytogenetic studies not only in instances with overt hematologic malignancies or complete myelodysplastic syndromes, but also when morphologic abnormalities occur in a single cell line.

Low megakaryocyte ploidy in Ph-positive chronic myelogenous leukemia measured by flow cytometry.

In chronic myeloproliferative disorders, the megakaryocytes differ in size and maturation compared with those of healthy individuals. In the present study, by using a 2-color flow cytometry technique, we determined the frequency of bone marrow megakaryocytes in different ploidy classes in 13 newly diagnosed and untreated patients with Philadelphia chromosome (Ph)-positive chronic myelogenous leukemia (CML) and in 12 healthy volunteers. The results showed a significant difference in megakaryocyte ploidy distributions between these 2 study groups. On the average, patients with CML had 59% of their megakaryocytes in ploidy classes 2N to 8N; in contrast, the healthy volunteers had only 22% of their megakaryocytes in classes 2N to 8N. Two patients with complex Ph translocation and 2 patients with a small clone with a chromosome abnormality in addition to Ph had the same ploidy distribution as those with only Ph translocation. The platelet count did not correlate with the megakaryocyte mean ploidy.

Cytogenetic studies in patients with mastocytosis.

Chromosomal aberrations in hematopoietic cells are common in malignant hematological disorders and have also been reported in some patients with mastocytosis. In this study, 34 patients with either urticaria pigmentosa or systemic mastocytosis were investigated by cytogenetic analysis of bone marrow cells. A follow-up investigation was performed in 22 patients. Clones with chromosome abnormalities were found in 32% of the patients at the first examination and in 27% at the second examination; in total, 41% of the patients had an abnormal clone in at least one examination. No clinical correlation was found with regard to cytogenetic results, with the exception of four patients who had an associated hematological disease and poor prognosis. In the second examination, only 6 patients had an unchanged chromosome pattern, and 4 of the patients with an initial normal pattern had appearance of abnormal clones; however, in 7 patients, the initial abnormal cells disappeared. The abnormalities were, among others, deletions of chromosomes 5, 7, 11, and 20. The proportion of cells with structural or numerical chromosome changes was higher in comparison with reported control groups. The frequency and type of chromosome abnormalities in bone marrow cells from patients with mastocytosis was about the same as observed in other chronic myeloproliferative disorders and myelodysplastic syndromes, diseases which also developed in 4 of our patients. An association between malignant hematological disorders and mastocytosis have been suggested by us and others. The chromosome abnormalities maybe reflect a genetic instability of the hematopoietic cells in mastocytosis.

Rearrangement of chromosome no. 3 in a case of preleukemia with thrombocytosis.

The clinical and cytogenetic findings of a patient with the preleukemic syndrome and a structural rearrangement involving both chromosomes No. 3 are described. The karyotypic abnormality consisted of an insertion of a part of the long arm of one chromosome No. 3 into the other, i.e., ins(3;3)(q27;q21q27). A prominent feature of the bone marrow was a marked megakaryocytic hyperplasia. The platelet count temporarily exceeded 1000 x 10(9)/liter. The findings of the present case, together with similar observations by others, suggest that the long arm of chromosome No. 3 may contain a region involved in the regulation of megakaryopoiesis.

Cytogenetic abnormalities in acute leukemia complicating melphalan-treated multiple myeloma.

The cytogenetic findings in 11 patients with multiple myeloma in whom a myelodysplastic syndrome or an acute nonlymphocytic leukemia developed are reported. All patients were treated with oral melphalan for 2-9 years in a total dose of 0.5-4.1 g. When examined during the myelodysplastic or leukemic phase, all patients had an abnormal bone marrow karyotype, hypodiploid in nine of the 11 cases. The chromosome abnormalities were clearly nonrandom and comprised a 5q deletion in three cases, monosomy 5 in four cases, deletion 7q--in two cases, and monosomy 7 in three cases. Loss of material from the long arm of chromosomes 5, 7, or both was found in eight patients. The different chromosome abnormalities were not associated with any specific morphological or clinical features.

Cytogenetic studies of bone marrow and extramedullary tissues and clinical course during metamorphosis of chronic myelocytic leukemia.

Of 33 consecutive patients with chronic myelocytic leukemia, examined during metamorphosis, 82% showed chromosome abnormalities in addition to the Ph1. Aberrations most frequently encountered were +8 (39%), +22q - (30%), and i(17q) (18%). Translocations other than the Ph1 were observed in four cases and - Y clones in four cases. Discrepancies in the cytogenetic pattern between bone marrow and extramedullary tissues or blood were noted in a total of 15 patients. In six cases, transformation occurred in extramedullary organs at a time when it was not present in the marrow. In three cases the bone marrow transformation was preceded by a lymph node blastic infiltrate; in one case, by a skin infiltrate; and in one case, by a subdural blastoma. Clonal abnormalities additional to the Ph1 were identified in the tumor tissue from all these cases. Patients with primary extramedullary transformation tended to have a lower median age at onset of metamorphosis, shorter survival, and higher incidence of chromosome abnormalities than the cases without extramedullary involvement. Patients with only Ph1-positive cells and no other anomalies had a slightly longer duration of metamorphosis and longer total survival. Basophilia and thrombocytopenia were more marked in cases with i(17q) than in the rest of the series.

Karyotypic evolution in Ph-positive chronic myeloid leukemia in relation to management and disease progression.

In a prospective study of 32 patients with chronic myeloid leukemia the frequency of chromosome abnormalities in addition to the Philadelphia chromosome (Ph) increased when the disease progressed. Before metamorphosis, 10 patients (31%) had developed additional abnormalities. Such abnormalities were present in three of them at the time of diagnosis; in the other seven, they were detected late in the chronic phase. New clonal abnormalities heralded or accompanied a more malignant phase of the disorder, usually a blastic leukemia. During metamorphosis, 78% of the patients had additional abnormalities, which in 68% of these cases comprised at least one of +8, +22q- or i(17q). Clones with additional abnormalities disappeared in eight cases, either spontaneously or in association with cytostatic therapy during the chronic or blastic phase. Involvement of chromosome #8, usually in the form of a trisomy, was found in 7 of 12 patients treated with busulfan, but was not found in any of the 10 hydroxyurea-treated patients, of whom 8 were splenectomized early during the chronic phase. Cells from the spleen, obtained by fine needle aspiration or splenectomy were cytogenetically examined in 18 cases during the chronic phase, but abnormalities in addition to the Ph were noted in only one patient, who was examined in the late chronic phase. The same abnormalities were present in bone marrow cells of this patient.

No increased frequency of trisomies 8 and 9 by fluorescence in situ hybridization in untreated patients with essential thrombocythemia.

Essential thrombocythemia (ET) is one of the diseases included among the myeloproliferative disorders in which trisomies for chromosomes 8 and 9 commonly occur. In ET, only a few patients are known to show clonal abnormalities. With fluorescence in situ hybridization (FISH), interphase cells can be evaluated and clones can be detected even though not revealed by conventional cytogenetic methods. By using FISH for enumeration of chromosomes 8 and 9 in bone marrow cells, we studied 22 patients with ET; 20 of them were investigated at the time of diagnosis when they were still untreated with myelosuppressive agents. Only two patients had trisomy 8; one of them was also found to have +8 with conventional cytogenetics. None of the patients had trisomy 9; two patients had borderline values in comparison to a control group. Thus, in ET, no increased frequency of patients with trisomy for 8 or 9 at the time of diagnosis could be detected with FISH.

Spontaneous remission during two pregnancies in a patient with essential thrombocythaemia.

A woman with untreated essential thrombocythaemia (ET) exhibited spontaneous normalization of her elevated platelet count during two pregnancies. After delivery the platelet count increased rapidly to the same high level as before. Low platelet adhesion as well as adrenalin- and collagen-induced platelet aggregation, present before pregnancy, increased to normal values during pregnancy. Furthermore, no erythropoietin-independent colony growth was observed during pregnancy, while such colonies were clearly demonstrated when the patient was assayed after pregnancy. This is the second case in the literature demonstrating that remission of ET can occur without treatment during pregnancy.

Bullous pyoderma gangrenosum in a patient with myelodysplastic syndrome during granulocyte colony-stimulating factor therapy.

An unusual case of bullous pyoderma gangrenosum in a patient with myelodysplastic syndrome during treatment with granulocyte colony-stimulating factor (G-CSF) is reported. The possible relationship between G-CSF therapy and pyoderma gangrenosum, as well as the beneficial effect of cyclosporin A therapy, is discussed.

Plasma erythropoietin concentrations in polycythaemia vera with special reference to myelosuppressive therapy.

In 80 patients with polycythaemia vera (PV) a total of 108 venous blood samples were obtained and analysed for EDTA-plasma erythropoietin (EPO) concentration. At the time of study 21 of the PV patients were newly diagnosed and had prior to blood sampling neither received phlebotomy treatment nor therapy with myelosuppressive agents; these subjects had a mean plasma EPO concentration of 0.5+/-0.9 IU/L. Thirty-seven patients treated with phlebotomy only had a mean plasma EPO concentration of 2.5+/-2.9 IU/L. The mean plasma EPO concentrations for 26 patients treated with hydroxyurea, 13 patients treated with radiophosphorous and 11 patients given a combination of myelosuppressive agents were 8.9+/-8.0, 10.9+/-12.6 and 7.2+/-7.4 IU/L, respectively. Untreated patients and patients on phlebotomy only had significantly lower values for plasma EPO than patients on therapy with myelosuppressive drugs. This finding persisted also after a correction for differences in haemoglobin levels had been introduced. Thereby, the present results would suggest a difference in the EPO feedback system in untreated and phlebotomised PV patients compared to PV patients treated with myelosuppressive agents.

Demonstrated Benefit of Continuous Interferon-Alpha-2b Therapy in Hairy Cell Leukemia. A Two-Year Follow-Up.

Interferon-alpha-2b (IFN) was given to a series of 50 patients with hairy cell leukemia (HCL). The IFN dose for both induction and maintenance was 2.0 × 10(6) IU/m(2) s.c. three times weekly. At 24 months 38 patients remained in the study. The proportion of complete responders (CR) increased during the follow-up, and had at 24 months reached 58%, while 28% at the same time had a partial (PR) and 14% a minor response (MR). During the two years of continuous IFN treatment none of the 38 patients showed any signs of relapse. The response rate was similar between splenectomized (n = 15) and non-splenectomized (n = 23) patients, but the rise in platelets was much steeper and reached a significantly higher plateau in patients, who previously had undergone splenectomy. The IFN therapy was generally well tolerated, but when evaluated at 24 months at least some (mostly mild) toxicity was noted in 76% of the patients. None of the patients developed neutralizing antibodies to IFN.

The bone marrow in urticaria pigmentosa and systemic mastocytosis. Cell composition and mast cell density in relation to urinary excretion of tele-methylimidazoleacetic acid.

The bone marrow sections from five normal subjects and 18 patients with mastocytosis were examined to establish criteria to distinguish urticaria pigmentosa from systemic mastocytosis. Nine patients had increased numbers of mast cells in bone marrow sections stained with a long toluidine blue staining technique specific for mast cells, whereas five patients exhibited increased numbers of mast cells on May-Grünwald-Giemsa-stained smears of bone marrow. A positive correlation between the number of mast cells in sections of the bone marrow and the urinary excretion of the main histamine metabolite tele-methylimidazoleacetic acid was found. In ten of the examined bone marrow specimens, focal lesions containing mast cells, lymphocytes, and eosinophils appeared. The presence of these focal lesions together with either an increased number of mast cells in bone marrow sections and/or increased urinary excretion of telemethylimidazoleacetic acid is considered diagnostic of systemic mastocytosis. No patient exhibited myeloproliferative condition or other major hematologic abnormality.

Increased serum erythropoietin concentration after allogeneic compared with autologous blood transfusion.

Serum erythropoietin (sEPO) level is known to increase as hemoglobin (Hb) concentration decreases during and after preoperative autologous blood donation (PAD). The endogenous erythropoietin (EPO) production after allogeneic blood transfusion has not to our knowledge, been studied. The aim of the present study was to determine whether there is, after surgery, any change in sEPO concentration after allogeneic blood transfusion, and whether there is any difference in EPO response after autologous or allogeneic blood transfusion. Thirty-one patients approaching total hip-joint replacement surgery, were randomized to receive either allogeneic red blood cells (n = 15) or predeposited autologous whole blood transfusion (n = 16). The relationship between Hb, sEPO, and reticulocytes in the recipients were repeatedly analyzed before, during and after surgery. The Hb followed an expected pattern, with a decreased concentration after PAD in the autologous group, then in both groups after surgery. The sEPO concentration was significantly higher in the allogeneic than in the autologous group on day one and day 4-5 postoperatively. The reticulocyte level, on the contrary, was higher in the autologous patients before, one hour after, and one day after surgery. The study showed a greater increase in sEPO concentration after allogeneic blood transfusion than after autologous blood transfusion. There may be an inverse relationship between sEPO and the reticulocyte level.

A prospective long-term cytogenetic study in polycythemia vera in relation to treatment and clinical course.

This paper reports the results of cytogenetic studies in a consecutive series of 64 patients with polycythemia vera, 57 of whom could be followed prospectively. The median length of the cytogenetic observation time was 93 months (range, 24 to 224 months) after diagnosis. Clonal chromosome abnormalities were observed initially in 11 patients (17%) and later during the course of the disease in another 20 patients. An abnormal karyotype was found in 71% to 80% of the patients who were examined after the development of myeloid metaplasia, myelofibrosis, or leukemia. Patients treated with myelosuppressive agents showed a significantly greater risk of chromosome abnormalities developing than did patients who had been phlebotomized. Acute leukemia developed in eight patients, all of whom had been treated with myelosuppressive agents. A chromosome abnormality preceded the leukemia in only two of the patients. The initial presence of an abnormal karyotype did not predict a greater risk of development of leukemia. No consistent relationship was demonstrated between the occurrence of chromosome abnormalities and the development of myeloid metaplasia and/or myelofibrosis, which was observed in 42% of the patients. The chromosome abnormalities followed a nonrandom pattern, and those most frequently observed were trisomies for 1 q, 8, 9, or 9p and deletion of 20q. Deletions seem to be common and were found in 14 patients.