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Bone marrow from 17 patients with refractory anaemia with excess of myeloblasts (RAEM) was cultured in methylcellulose semi-solid medium. Compared with normal bone marrow, 3 patterns of growth occurred corresponding with different clinical stages of the condition. Patients whose bone marrow grew normal colonies and those who produced a predominance of microclusters had the longest life expectancy, while those who produced a predominance of macroclusters had the shortest life expectancy with a high rate of acute leukaemic transformation. Colony culture appears to be a useful prognostic tool in this condition.

Cell kinetics have been studied at several periods in a patient with typical cyclic neutropenia. Peripheral blood granulocyte labelling showed an increased destruction in the preleucopenic phase. Bone marrow colony-forming cells and blood leucocyte colony forming activity were normal or above the normal range. The major abnormalities were found in sequential studies of bone marrow proliferation measured by "in vitro" 3H-thymidine flash labelling. A fall in the labelling index of promyelocytes was observed in the second part of the cycle. Incubation of normal human bone marrow cells with patient's granulocytes showed a marked decrease in 3H-thymidine incorporation, compared with incubation with the same number of normal granulocytes. Human cyclic neutropenia seems to be due to a factor secreted by abnormal polymorphonuclears inhibiting myeloid proliferation.

Over 20 years, 58 cases of PV in young people (46 meeting the full PVSG criteria, 12 with elevated red cell volume and leucocytosis or thrombocytosis, without splenomegaly) were studied and have been followed for periods of 3-24 years. These cases represent approximately 5% of the cases of PV referred to the Department of Nuclear Medicine of St Louis Hospital during this period. They differ from older patients in the initial clinical severity, the short interval between the first symptoms and the diagnosis, frequent presentation with a life-threatening complication (two cases of hepatic vein thrombosis, six thrombotic or haemorrhagic events, six splenectomies, two abortions) and a very enlarged spleen in half the cases. However, after the initial complications, the overall survival is very long (exceeding 70%, even when including the initial complications, at 15 years). The vascular accidents occur exclusively in the phlebotomized patients, the main risk factor being the poor stability of the haematocrit. Only one acute leukaemia was observed among the 14 cases treated by radioactive phosphorus and/or alkylating chemotherapy. The most frequent late complication was evolution towards myelofibrosis. This spent phase seemed to occur earlier in patients treated by phlebotomy. On the basis of this data, we would advise the following therapeutic strategy: phlebotomies, as soon as the diagnosis is established, and a systematic long-term treatment by hydroxyurea with the hope of reducing the number of vascular complications and of delaying the evolution towards the spent phase and the myelofibrosis.

An analysis of the risk of progression towards leukemia, carcinoma and myelofibrosis was performed in 93 patients treated by 32P alone (PVSG protocols) since 1970-1979, 395 patients over the age of 65 years treated by 32P with or without maintenance therapy using hydroxyurea (French protocol) since 1980-1994, and 202 patients under the age of 65 treated by either hydroxyurea or pipobroman since 1980. The risk of leukemia, or myelodysplasia, or lymphoma in the 32P-treated patients was 10% at the 10th year, but increase after that time to reach a value of about 30% at the 20th year, in the surviving case. This risk was not dose-related. Despite a marked reduction of the cumulative 32P dose in the patients maintained by hydroxyurea, the actuarial risk was 19% at the 10th year. In the patients treated exclusively by non radio-mimetic agents (hydroxyurea or pipobroman) a risk of 10% at the 10th year was observed. The risk of carcinoma (excluding skin cancers) was about 15% at the 10th year in the 32P-treated cases, a value similar to that generally reported by the French statistics. There was no prevalence of digestive carcinomas. In contrast, the patients receiving 32P and hydroxyurea as maintenance had an excess risk: 29% at the 10th year. In the relatively young cases treated by non radio-mimetic agents, the risk was similar in both arms: 9% at the 10th year, similar to the expected incidence at this age. The risk of myelofibrosis with myeloid metaplasia was still relatively low at the 10th year, about 15% in all arms, but increased towards a value higher than 30% in the patients surviving at the 20th year. At the present time, but in only a few cases with long-term following, no myelo-fibrosis with splenic metaplasia has been observed in the pipobroman-treated cases. The present results, which need to be confirmed (the present analysis has been done in spring 95) suggest that:-the use of non radio-mimetic agents does not protect against leukemic transformation, which may be a consequence of the disease; rather than of the treatment,-maintenance therapy after initial use of 32P increases the risk of both leukemia and carcinoma,-and hydroxyurea does not delay the risk of developing myelo-fibrosis, in comparison with 32P alone.

The very-long-term follow-up of patients initially included in the PVSG protocols provides useful information. The excess risk of cancer after chlorambucil appears to persist for 5 years after stopping this treatment. The risk of leukaemia induced by marrow suppression (32P or chemotherapy) was marked before the 10th year, but low thereafter. Phlebotomy is unacceptable as permanent treatment because of the poor clinical tolerance and the frequency of vascular complications. This treatment is also associated with a risk of early progression towards myelofibrosis with myeloid splenomegaly. In the very long term, 15 years or more after the diagnosis, this complication is the major clinical risk, affecting almost 50% of our patients surviving at this time. The prevention of this type of complication could constitute one of the objectives of future protocols dealing with this disease.

In the new context of the use of retinoic acid (RA) therapy as an inducer of leukemic differentiation and a selective inhibitor of human myeloid leukemia cell growth, we undertook to explore the potential physiological role of retinoids on the proliferation and differentiation of normal bone marrow myeloid progenitors. The effects of continuous exposure of all-trans-RA, its naturally occurring isomer, 13-cis-RA, and its metabolite 4-oxo-all-trans-RA were studied on the growth of normal human bone marrow cells in soft agar, directly and after liquid culture. Retinoids enhanced the total number of granulocytic colony and macrocluster formation in the presence of exogenous colony-stimulating factor (n = 9). Dose-response curve were bell-shaped, with a maximal increment between concentration of 0.5 and 0.05 nM. In all cases, a concomitant decrease of macrophagic colonies was noted. The positive effect on granulocytic colony formation was observed with each of the retinoids tested (all-trans, 13-cis and 4-oxo-all-trans) (n = 5). On erythroid colony formation, all-trans-RA had the opposite effect. Constant suppression of CFU-E and BFU-E colony formation and coloring was observed in a dose-related fashion from 0.1 to 10 microM (n = 5). Thus, in granulocytic, as in erythroid colony formation, retinoids affected both proliferation and differentiation parameters. However, after short-term suspension culture in the presence of all-trans-RA, an increase of both CFU-GM and BFU-E colonies, was observed. These results suggest a specific effect of retinoids on late myeloid precursors and places retinoids as possible candidates for enhancement of normal granulocytic differentiation.

Teh technical conditions for surface counting and the methods for splenic and liver sequestration after injection of 51Cr-labelled cells were studied in 96 patients. A comparative study of different sites of positioning of the detection probes leads us to recommend the mid-line at the third intercostal space for the precordial area, the point of maximum count-rate obtained at each measurement for the splenic area, with the patient in right lateral decubitus position and the probe vertical, and a point situated on the midclavicular line 4 cm above the right costal margin for the liver area. A comparative study of different methods of calculation leads us to recommend the method known as 'excess counts', but with a correction of the gross values based on the evolution of circulating radioactivity and not of the count-rate measured over the precordial area. Measurement of urinary excretion of radioactivity showed that only a minor part was due to the loss of the tracer from the sequestration sites, and that the level varied only slightly between subjects. Thus the loss of tracer does not interfere with the interpretation of external counting data. Much of the disagreement regarding interpretation of the results of external counting in clinical practice is due to technical problems. Standardization of techniques of measurement and interpretation of results, selecting those which this study indicates as the most reliable, would permit better exchange of information between laboratories and clearer conclusions as to the practical use of surface counting.

Fifty-one cases of pure, primary erythrocytosis were identified and followed at Hopital Saint-Louis, Paris, and compared with 350 cases of polycythemia vera (PV) observed during the same period. At the initial evaluation, these cases did not differ from PV cases with respect to age, sex ratio, degree of red cell volume increase, and clinical symptoms. They did differ by the absence of splenomegaly, granulocytosis and thrombocytosis. At a late stage of evolution only a few cases developed classical criteria of PV. From this group of apparently homogeneous cases, two subgroups evolved. Sixty percent of the cases were highly responsive to myelosuppression with /sup 32/P. The median duration of the first remission was greater than five years, the mean yearly dose of /sup 32/P was very low, and there was a low incidence of complications. The other group (40% of cases) was relatively resistant to myelosuppressive agents. The development of better methods of investigate this disorder might help in discriminating these two groups from both an etiological and pathophysiological viewpoint. The thromboembolic risk of these diseases suggests that myelosuppressive therapy should be utilized in older patients with higher risk of vascular accidents, reserving phlebotomy for younger patients and those who aremore » shown to be resistant to /sup 32/P therapy.« less

Simultaneous bone marrow scintigraphy with 99m Technetium colloids and 111 Indium transferrin was performed on 34 cases of preleukemic anemias and was shown to be of good prognostic value. Groups of different outcome were defined: for a normal and parallel uptake of the two markers, 90% of the patients died of acute leukemia; for a low Indium and high Technetium uptake, only 1 patient out of 15 died of leukemia (P less than 0.001). Standard clinical and hematological data were of no predictive value. Iron kinetic data and CFU/GM colony growth were correlated to the scintigraphic results. Taken together, these three kinetic parameters have a good sensitivity and specificity for the prognosis of preleukemic states.

Although abnormalities in granulopoiesis detected by means of bone marrow cytology, culture and kinetic studies have provided prognostic data in preleukemic states and oligoblastic leukemias, this information cannot be applied to individual cases. In order to determine the indications for treatment and the form it should take in a given case, data would be required concerning the probability of impending transformation into acute leukemia. In 45 studies involving 34 patients who were followed for 10–42 months, a combination of a rise in the proportion of granulocyte precursors in S-phase, indicated by a colony-forming cell suicide rate of over 40%, and a low labelling index of myeloblasts and promyelocytes, was always followed by the onset of acute leukemia within 10 months. Sequential studies in 13 patients revealed an increase in clusterforming cells and in the suicide level in the second study. The changed kinetics of granulopoietic proliferation may provide an indication for chemotherapy.

The analysis of 288 cases of polycythemia vera (PV) with a minimal follow-up of 10 years enabled us to study the characteristics of acute leukemia as observed in 33 patients (11.4%). In 50% of the patients (16 of 33), the malignant transformation is of the refractory anemia with excess of blasts (RAEB) type. Half of these further transform to acute nonlymphocytic leukemia (ANLL). Their life expectancy is not better than patients who abruptly develop ANLL. Leukemic transformation shows a frequency peak in the eighth year after initial evaluation in PV treated with chemotherapy and in the 11th year in patients treated with radiotherapy. In 30% of the patients myelofibrosis, or the spent phase of PV, is present before the transformation to acute leukemia (AL). This complication is, however, part of the natural history of PV and is observed in 20% of PV patients at 10 years when leukemic transformation is absent. Marrow fibrosis can therefore not be considered as a preleukemic phase. It was also noted that the occurrence of myeloid metaplasia/myelofibrosis is more frequent and begins earlier in patients treated by phlebotomy alone, and who do not transform to leukemia. The clinical characteristics of these AL, including high frequency of partial marrow invasion, difficulties in cytologic classification, a peak incidence similar to that in patients treated by chemotherapy or radiotherapy for a prior malignancy, multiple chromosome abnormalities, and poor response to therapy are all highly suggestive of secondary leukemias.

Summary. In vivo kinetic studies of granulocytes labelled in vitro with 51 Cr and DF32P were carried out in nine haematologically normal subjects by isolation of the cells in the blood samples by the Ficoll-Isopaque flotation method. 51 Cr and 32P specific activity of blood samples made of 93–98% granulocytes was studied. Distribution between marginated and circulating granulocyte pools was identical for both labelled cells and the marginated pool was similar to the circulating pool, except that it was lower in one subject who had a previous splenectomy. The half-disappearance time (T 1/2) was 16.1 ± 2.2 h for 51 Cr-labelled and 5.4±2.1 hr for DF32P-labelled granulocytes. In one case of a normal subject who previously received multiple transfusion homologous 51 Cr-labelled granulocytes had a T1/2 of less than I h.

The inhibitory activity of peripheral blood lymphocytes on autologous bone marrow was studied in 27 patients with aplastic anaemia after treatment with androgen. Inhibitory activity was hard to assess in 10 patients studied during the first year of treatment. The colony count was too low to be certain of differences between the samples incubated with or without lymphocytes. Among the 17 patients who had more than 10 colonies per 2 x 10(5) mononuclear bone marrow cells, nine showed inhibitory activity by peripheral blood lymphocytes. After 12 months of androgen therapy each of these patients showing inhibitory activity of bone marrow colony forming cells by peripheral lymphocytes responded to antithymocyte globulin. None of nine patients with few colony forming cells or no inhibitory activity of lymphocytes responded to immunosuppression.

One hundred thirty-five polycythemia vera (PV) patients (30 untreated by chemotherapy and 105 treated) were studied cytogentically. The incidence of clonal chromosomal abnormalities was 20.7% (28 patients in nonleukemic phase). The incidence of 20q− was 3.7% (5 patients). The presence of cytogenetically abnormal clones did not allow prediction of the evolution of the disease. In a few cases, abnormal clones disappeared at the time of later studies. Although nonrandom, the majority of clonal chromosomal abnormalities are believed to be secondary events in PV patients.

Bone marrow granulocyte colony forming cells (CFU-C) were measured in 10 HL'A compatible sibling donor-recipient pairs. The regeneration of granulocytes and CFU-C compartments were also studied in order to evaluate haemopoietic recovery. The number of nucleated bone marrow cells in the donation was 23 ±4 times 109 cells, which recipients received (3.5 ±0.4) × 108 nucleated cells/kg and (1.19 ±0.32) × 105 CFU-C/kg. This produced a bone marrow reconstitution of (67 ±26) × 105 CFU-C/kg by day 30. There was a significant correlation between CFU-C/kg given and (1) granulocyte count on day 30 (P = < 0.05) and (2) the first day of reappearance of neutrophils in the blood (P = < 0.05). a significant correlation between CFU-C/kg given and (1) granulocyte count on day 30 (P = < 0.05) and (2) the first day of reappearance of neutrophils in the blood (P = < 0.05). These results indicate that the speed and completeness of granulocyte regeneration can be predicted by measurement of the size of the CFU-C inoculant in the bone marrow graft.

A total of 19 cases of chronic refractory anaemia underwent simultaneous in vivo erythrokinetic study and in vitro bone marrow culture. They were followed up clinically for at least 2 years. Good correlation has been found between erythrokinetic data (simultaneous quantitative and qualitative disorders of erythroblastic proliferation in preleukaemic states; pure qualitative disorders in primary sideroblastic anaemia) and the results of culture of granulocyte precursors in the bone marrow (small number of colonies, reduced size of colonies in preleukaemic states; normal number and growth in non malignant refractory anaemia). It would seem thus that both examinations are of practical interest in clinical haematology, making it possible to foresee the malignant evolution of some refractory anaemias.

Chromosome studies were performed on 15 patients suffering from acute nonlymphocytic leukemia (ANLL) and in one patient in a preleukemic state following polycythemia vera (PV). Clonal chromosome abnormalities that were present in all cases were clearly nonrandom and involved chromosomes #1, #5, #7, #8, #9, #11, and #21. A subdivision of ANLL into two categories occuring in the course of PV is proposed from the clinical, hematologic, and cytogenetic data: one resembling de novo ANLL with rapid initial evolution, easy classification into one group of the FAB nomenclature, and simple chromosome abnormalities; the other resembling induced leukemia, often with more progressive initial evolution, difficulty or impossibility of classification into one group of the FAB nomenclature, and complex chromosome abnormalities. The consequences for the committment level of progenitor cell from which the leukemic clones originate are discussed.

A clinical phase (spent phase) in the course of polycythaemia vera (PV) cases is described as enlargement of the spleen in spite of treatment, frequent cytopenia of one or several lines, persistent red cell hypervolaemia with considerable increase of plasma volume, persistence of myeloid hyperplasia with no collagen myelofibrosis or osteomyelosclerosis, absence of hepatosplenic erythroblastic metaplasia, as shown by radio-iron kinetics and/or 111In-transferrin scintigraphy. The frequency of this phase was 5% in a study where it was not systematically sought, but it could in fact be greater. Its occurrence is not related to the clinical and biological parameters of PV. On the other hand, it is significantly more frequent and earlier in patients treated by phlebotomies than in those treated by myelosuppression (32P). In four of the 12 cases, this phase was rapidly followed by an acute leukaemia. In eight cases there was a 1-5 year interval before a myelofibrosis with splenic myeloid metaplasia. This evolution could at this stage be delayed by chemotherapy. The efficacy of splenectomy should be studied.

Eighty-three patients with myelofibrosis have been studied by erythrokinetics and have been followed up until death or for at least 12 months. Because of a large plasma volume the venous haematocrit gives only a poor idea of the red blood cell volume. The red cell survival was reduced in the majority of cases but significant haemolysis was rare. The amount of haemolysis of autologous and isologous red cells was similar, suggesting an extra-corpuscular origin for the haemolysis. Plasma iron turnover was always increased, sometimes markedly, but red cell iron incorporation was reduced in 70% of cases, indicating ineffective erythropoiesis. Surface counting showed an absence on diminution of sacral iron fixation and a rapid and marked splenic uptake in more than 90% of the cases; uptake of iron by the liver occurred in half the cases, usually not very high; iron release from the spleen was absent or reduced in 67% of the cases. The degree of ineffective erythropoiesis as measured by radio-iron incorporation and release by the spleen, the amount of haemolysis, and the red cell volume were strongly correlated with prognosis. These factors enabled a more precise prediction to be made of the clinical outcome in the 2 years following the study, than the clinical data alone. A prospective study might show whether erythrokinetic studies are also useful in determining the choice of treatment.

Thirty-four patients with polycythemia vera complicated by pruritus were treated with 900 mg of cimetidine daily for 30 days and their responses to treatment were evaluated. The conditions of 15 (44%) were improved, with 12 patients stating that pruritus completely disappeared. Nineteen patients either showed no improvement or had increasing symptoms. No toxic effects were reported. The positive responses seen are encouraging and suggest that controlled studies are indicated to further evaluate the effectiveness of H2antagonists. (Arch Intern Med1982;142:241-242)

Cross-presentation is an important mechanism to elicit both immune defenses and tolerance. Although only a few DC subsets possess the machinery required for cross-presentation, little is known about differences in cross-presenting capabilities of DCs belonging to the same subpopulation but localized in different lymphoid organs. In this study, we demonstrate that steady-state thymic CD8(+) DCs can efficiently cross-prime naïve CD8(+) T cells in the absence of costimulation. Surprisingly, cross-priming by splenic CD8(+) DCs was dependent on licensing factors such as GM-CSF. In the absence of GM-CSF, antigen-MHC-class-I complexes were detected on thymic but not on splenic CD8(+) DCs, indicating that the cross-presentation capacity of the thymic subpopulation was higher. The observed cross-priming differences between thymic and splenic CD8(+) DCs did not correlate with differential antigen capture or costimulatory molecules found on the surface of DCs. Moreover, we did not detect overall impairment of antigen presentation, as peptide-loaded splenic CD8(+) DCs were able to induce CD8(+) T-cell proliferation. The observation that thymic CD8(+) DCs are more efficient than splenic CD8(+) DCs in T-cell cross-priming in the absence of licensing factors indicates that the requirements for efficient antigen presentation differ between these cells.