Diagnostic and prognostic value of DNA image cytometry in myelodysplasia.

The DNA content of erythropoietic cells from 10 patients with refractory anaemia (RA) with megaloblastic changes, who subsequently developed acute non-lymphoblastic leukaemia (ANL), and from seven patients with megaloblastic marrow aspirates due to pernicious anaemia were compared by DNA image cytometry. The DNA distribution, the rate of aneuploid cells exceeding 5c (5cER), and the square deviation index of DNA values from the normal 2c-peak (2cDI) were recorded. Both variables were of diagnostic and prognostic importance for epithelial tumours, malignant lymphomas, and dysplastic lesions. A rate of 5cER greater than 0 was found in eight of 10 myelodysplastic, but in none of seven control cases. Hypodiploidy was equally pronounced in both groups of patients. The 5cE had the highest discriminative value of all variables calculated. The 2cDI was not significantly different in either group. In pernicious anaemia the 2cDI depended mainly on the percentage of S cells, reflecting the defect of DNA synthesis. In RA with megaloblastosis the 2cDI correlated with the percentage of G2 cells, reflecting G2 arrest. In the myelodysplastic group the 2cDI correlated positively with the length of time until ANL developed, indicating the prognostic relevance of 2cDI. Our findings show that in megaloblastic anaemia DNA image cytometry can distinguish myelodysplasia from pernicious anaemia and that it also provides prognostic information.

Morphometric assessment of bone marrow fiber content in acute nonlymphatic leukemia at presentation.

The number of intersections of reticulin fibers per sq mm of fat cell-free marrow parenchyma with the lines of a grid ocular (i/sq mm) represents an objective measure of the bone marrow reticulin fiber content. This method was used to assess the reticulin fiber content of bone marrow biopsies from 50 cases of acute nonlymphatic leukemia (ANLL) at presentation and 20 controls. Seventeen (34%) of the 50 patients with ANLL showed fibrosis, i.e., had a reticulin fiber score above the upper 99% confidence limit of the mean of 20 normal control biopsies. The frequencies of marrow fibrosis, as defined above, were 47% (16 of 34) in the combined subtypes of undifferentiated (M0), myeloid (M1), myelomonocytic (M4) and monocytic (M5) acute leukemia and 7% (1 of 15) in the combined subtypes of acute myeloid leukemia with partial maturation (M2) and acute promyelocytic leukemia (M3) (P less than .01). The fibrosis scores of M0/M1/M4/M5 patients were significantly higher than those of M2/M3 patients (P less than .05) and of controls (P less than .005). Finally, the survival of patients with and without fibrosis was not different.

The prognostic implication of clinical and histological features in Ph1+ chronic myelocytic leukaemia (CML).

A study on clinical and histological features of prognostic significance was performed in 45 patients with Ph1+ -CML who showed median survival of 36 months. The histological features were evaluated by morphometry of iliac crest biopsies. Among the variables correlated with prognosis, we eliminated those without primary importance by mutual univariate retrospective stratification. Thus a clinical and a histological set of important prognostic criteria was established. Their influence on prognosis proved to be independent of each other and therefore could be used for separate classifications. Clinical classification yielded two groups with different prognosis: compared to the rest of the patients, the prognosis was much worse for those with a spleen size greater than 10 cm, a liver size greater than 2 cm (below costal margin) and greater than 5% circulating blasts plus promyelocytes. The histomorphological classification consisted of three subgroups: a better than average prognosis was found for patients with pseudo-Gaucher cells in the bone marrow, while in the remaining cases the prognosis was worse in patients with a high number of megakaryocytes (greater than 70/mm2) and a low volume ratio of granulopoiesis: megakaryocytes (less than 15). Since liver size was correlated with the duration of prediagnostic symptoms, the clinical classification probably reflects different disease stages, i.e. a later CML diagnosis. However, the histological set of prognostic factors is independent of the length of the prediagnostic period. Consequently, this morphological classification seems to discriminate different subgroups. Another important prognostic factor, marrow fibrosis, was independent of other histomorphological features, and correlated with duration of symptoms. It obviously also indicates more advanced disease.

[Myelosarcoma as the primary manifestation of acute myeloid leukemia]. / Das Myelosarkom als Primärmanifestation einer akuten myeloischen Leukämie.

Myelosarcoma ("Granulocytic sarcoma", "Chloroma") is an extramedullary tumor composed of granulocytic precursor cells and related to myelogenous leukemia. If the tumor precedes acute leukemia diagnosis is difficult and requires special diagnostic techniques. This is documented by the presented 7.5 years old girl with primary myelosarcoma. In spite of early and intensive chemotherapy and radiation the sarcoma soon was followed by acute myelogenous leukemia with skin infiltrations. Cytogenetic classifications may in future lead to the development of a differentiated therapy of myelosarcoma.

Recovery patterns of rat hemopoietic stem cells between pulse doses of 7,12-dimethylbenz(a)anthracene (DMBA) applied in a leukemogenic regimen.

Male Wistar rats received repeated pulse doses of 7,12-dimethylbenz(a)anthracene (DMBA), known to elicit myelodysplasia followed by acute, mostly erythroblastic, leukemia at 10-day intervals. The recovery of spleen colony forming hemopoietic stem cells (CFU-s) surviving the cytocidal action of DMBA was examined between pulses. Recovery after a pulse of 35 mg/kg body weight varied with the organ source of the CFU-s (femoral bone marrow or spleen) and the number of preceding DMBA pulses. After a single DMBA pulse bone marrow CFU-s initially recovered faster than reported for normal bone marrow CFU-s transplanted into chemically conditioned rats. But recovery was followed by regeneration arrest. Population doubling times of marrow CFU-s increased with the number of DMBA pulses. Recovery of splenic CFU-s was slower after a single DMBA pulse than reported for normal spleen CFU-s transplanted into chemically conditioned rats. The CFU-s population doubling times were not significantly different after a single or five DMBA pulses. After three pulses, however, recovery of splenic CFU-s was exceedingly slow until day 5 and subsequently accelerated, but was still slower than after one or five pulses. In the spleen CFU-s recovery was always accompanied by regeneration of total cell numbers with a preference for erythroid regeneration. In the bone marrow this was the case after three DMBA pulses only.

Acute megakaryocytic myelosis preceded by myelodysplasia. Report of a case and review of the literature.

Acute megakaryocytic myelosis represents a distinct disease entity. As shown by a survey of the literature, it is a rapidly fatal disease, not preceded by a chronic myeloproliferative disorder, and mostly refractory to cytotoxic treatment. The first manifestation is pancytopenia with initial absence but quick development of hepatosplenomegaly. In contrast to "acute myelofibrosis", which is characterized by hyperplasia of all three haematopoietic lineages, a purely megakaryocytic proliferation develops together with a slight reticulinic fibrosis. Immature megakaryocytic cells may appear in the circulation, allowing classification of the disease as a variant of acute non-lymphatic leukaemia. This view is stressed by the observation of a preceding myelodysplastic phase in the case reported here.

Aplastic anaemia and the hypocellular myelodysplastic syndrome: histomorphological, diagnostic, and prognostic features.

In a retrospective study of 111 patients with aplastic anaemia iliac crest biopsies were evaluated for the presence of morphological features statistically related to the evolution of the disease. Prognostic variables for a transition to acute non-lymphatic leukaemia were: cellular atypias of the three haemopoietic lineages, as observed in the myelodysplastic syndrome, and especially "micromegakaryocytes"; high numbers or irregular distribution of megakaryocytes, or both; and (slight) marrow fibrosis. Clinical variables did not influence these prognostic correlations. Prognosis in relation to death from bone marrow failure without leukaemia might well have been influenced by a strong plasma cell reaction, but this correlation was weakened by clinical factors. On the basis of this study aplastic anaemia can thus be subdivided morphologically into two disease entities--namely, hypocellular myelodysplastic syndrome with a 23-82% risk of acute non-lymphatic leukaemia developing within three years, depending on how many variables associated with acute non-lymphatic leukaemia are present, and non-dysplastic myelohypoplasia.

Preleukemic myelodysplastic syndromes (MDS): pathogenetical considerations based on retrospective clinicomorphological sequential studies.

Analysis of myelodysplastic syndromes (MDS) in 77 patients terminating in "overt" leukemia revealed sequential changes of marrow morphology with respect to cellularity and involvement of lineages, indicating that different "forms" of MDS represent in reality different phases. Three main phases were observed, which occurred in the following non-reversible sequence: (a) hypocellular dysplasia (H), (b) hypercellular dysplasia with predominance of erythropoiesis (E), and (c) with predominance of myelo(mono)poiesis (MM). Published studies suggest that these phases represent different manifestation stages of the stem cell lesion leading to MDS. Manifestation may probably be promoted by factors causing marrow aplasia. Transition to "overt" leukemia in several cases occurred locally, not throughout the whole marrow. In some patients a mature myelo(mono)-cytic cell population showed the capacity for widespread tissue infiltration, characteristic of leukemic cells. This argues against the theory that "overt" leukemia is the result of continuous dedifferentiation in MDS, but favours the concept of multiple initiating "events" leading to evolution of a leukemic subclone within a myelodysplastic clone. A connection between leukemia differentiation and MDS phase, from which leukemia developed, was also observed indicating that the risk of different stem cell subpopulations to become the target of the leukemia initiating "event" varies during the course of myelodysplasia.

Bone marrow mast cell reaction in preleukaemic myelodysplasia and in aplastic anaemia.

The relationship of bone marrow mast cell counts to prognosis was investigated in 48 patients with preleukaemic myelodysplasia, in 59 patients with aplastic anemia and in a DMBA induced myelodysplasia/leukaemia rat model. In patients with myelodysplasia terminating in overt leukaemia the number of mast cells per square millimeter was not correlated to duration of the preleukaemic course. Leukaemia development probabilities of patients at risk were not different for low and elevated mast cell counts. In aplastic anaemia, however, a lower bone marrow mast cell count was related to a higher survival probability and longer survival time. In the animal model no significant differences could be found between myelodysplastic, leukaemic, and control animals.

[Cryotherapy of rectal cancer. Immunologic results]. / Zur Kryotherapie des Rektumkarzinoms. Immunologische Ergebnisse.

On the basis of experimental studies carried out in animals several authors could demonstrate that cryotherapy is able to induce a raise of auto- and tumour-antibodies. The specificity of these reactions has been proved. Comparative analysis could verify greater immune response after 'in situ-destruction' of tumours than after conventional surgical excision. In 13 patients cryosurgically treated for inoperable rectal carcinoma a raised concentration of IgG and IgA could be shown in several patients in the area surrounding the carcinomatous cells by means of a direct immune fluorescent technique in biopsies of the tumour. Immune globulines of the class IgA seemed to be important. Our results suggest a probable correlation of immune response and clinical course. The prognostic value of our method has to be examined in a larger group of patients and laboratory experiments. Cryotherapy can be regarded as one of several possible forms of treatment to brake the barrier of immunity in cancer patients.

The possibility of assaying Wistar rat bone marrow CFUs in a xenogeneic (rat-to-mouse) system.

The possibility of replacing the space-consuming rat-to-rat colony-forming unit (CFUs) assay by rat-to-mouse assay systems was examined using Wistar rat bone marrow. After considering the published results on the responsiveness of mouse strains to hemopoietic xenografts and on the ways to abrogate "xenogeneic resistance', we tested C57B1/6J and C3H/He mice conditioned by cyclophosphamide (CY) and/or whole-body irradiation in the following combinations: 850 rad C57Bl; 850 rad + CY C57Bl; 800 rad + CY C3H. A linear relationship between the number of cells injected and the macroscopical spleen colony count could be demonstrated with all three combinations. However, we observed a high number of endogenous colonies in the 850 rad C57B1 system. The results were confirmed by karyotype analysis. Colony yield and seeding efficiency with 800 rad + CY C3H were comparable to the rat-to-rat assay, but were considerably lower in the case of 850 rad + CY C57B1. In the latter system, the colonies were primarily erythroid.

S-phase resistance of rat hematopoietic stem cells to 7,12-dimethylbenz(a)anthracene cytocide and its implications for leukemia development.

In the experimental rat leukemia system, induced by repeated 7,12-dimethylbenz(a)anthracene (DMBA) pulses, the sensitivity of the spleen colony-forming hematopoietic stem cells (CFU-s) to the cytocidal action of a challenging DMBA injection (35 mg/kg body weight) varied with the number of pulses already applied and the organ source of CFU-s (femoral bone marrow or spleen). Assessment of the fraction of DNA-synthesizing CFU-s with the [3H]thymidine suicide technique at the time of DMBA challenge and comparison with the 20-hr CFU-s reduction values by DMBA in vivo showed an inverse correlation (p less than 0.001). It was deduced, therefore, that S-phase CFU-s are relatively resistant to DMBA cytocide. Since initiation by chemical carcinogens has been shown to be relatively S-phase specific, S-phase-resistant cytocide would lead to a selection of initiated cells and, in the case of repeated applications, to a selection of cells with multiple successive initiation hits. Preferential differentiation and organ site of leukemia, as well as evolution in sequential morphological steps, fit this assumption.

On the pathogenesis of preleukemic myelodysplastic syndromes: development of a dysplastic hemopoietic proliferation in the rat after a single pulse dose of dimethylbenz(a)anthracene (DMBA).

After a single pulse dose of DMBA, rats develop bone-marrow hypoplasia, which is almost compensated for by regeneration after 16 weeks. Subsequently, dysplastic signs of hemopoiesis appear in all experimental animals as massive extrusion of normoblasts into the peripheral blood, red-cell aniso- and poikilocytosis, nuclear deformities, atypical mitoses, and PAS-positivity, as well as megaloblastoid maturation dissociation of erythroblasts and nuclear and granulation anomalies of neutrophilic granulocytes and monocytes, comparable to human "pseudo-Pelger cells" and "paraneutrophils". At the time of death (112-497 days after DMBA pulse) experimental animals showed hyperplastic bone marrow with increased granulopoietic/erythropoietic ratios and an augmented, mainly erythropoietic, hemopoiesis in the spleen, with splenomegaly in six rats. Splenic hemopoiesis is accompanied by white pulp atrophia. The cause of death was septicopyemia in three rats, anemia in three, and bleeding in one rat. None of the animals developed a leukemic blast phase. Myelodysplastic changes in this experiment are the same as have been shown to precede leukemia in rats treated with five DMBA pulses (Fohlmeister et al. 1981). Possible relations of myelodysplasia and leukemia are discussed.

[Hypoferric anaemia due to a leiomyosarcoma of the proximal jejunum (author's transl)]. / Eisenmangelanämie durch Leiomyosarkom des proximalen Jejunum.

In an outpatient the diagnosis of a hypoferric anaemia and a transitory, masked, intestinal bleeding led to the discovery of a tumour in the proximal jejunum. The diagnosis was confirmed by operation. Radiographs and surgical specimen corresponded to each other. The exact diagnosis leiomyosarcoma could only be made histologically. Sarcomata of the proximal jejunum are extremely rare.