Paul C. Vincent

Towards a cure of acute granulocytic leukemia

Abstract This paper reviews progress in the therapy of acute granulocytic leukemia (AGL) and examines reasons why its results have remained unsatisfactory. For some years there has been no distinct improvement in remission rates, and survival has not lengthened greatly in most patients attaining remissions. However, in a few patients survival is of considerable length. Improvements in supportive therapy have helped raise remission rates in older patients and in those with high leukocyte counts. Resistance to induction therapy is now mainly due to intrinsic resistance of leukemic cells whose growth kinetics are such that only a minority can respond to cycle-specific therapy. Attempts to manipulate the cell cycle have not so far increased remission rates. No clear evidence exists that maintenance therapy prolongs remissions. Relapses are almost certainly due to re-growth of suppressed leukemic clones. Among new forms of treatment, immunotherapy is not yet securely based. Marrow transplantation may become occasionally useful. It is questionable if cytotoxic therapy alone will improve the prognosis of AGL radically. Though there is little to show that progress towards a cure is being made, new approaches based on a better understanding of the pathology of leukemic growth may eventually be translated into clinical success.

Studies on muramidase in hematologic disorders. I. Serum Muramidase and Serum Lactic Dehydrogenase in Leukemia

Estimations of serum muramidase (S.M.) and lactic acid dehydrogenase (L.D.H.) were made at the time of presentation in 79 adults with various forms of leukemia and compared with a number of other biochemical and hematologic parameters. In 35 patients, serial studies of these enzymes were also carried out. Initial S.M. levels, but not L.D.H. levels, were found to be of assistance in the diagnosis of the type of leukemia present. In patients with acute myeloblastic leukemia (A.M.L.), high pretreatment levels of S.M. tended to have a favorable prognosis. There was good correlation between initial S.M. and L.D.H., serum uric acid, total white cell count, absolute blast and granulocyte counts when all leukemias were taken together. However, this was not a consistent finding in analyses of the individual types of leukemia. In serial studies, S.M., and to a lesser extent L.D.H., paralleled the hematologic fluctuations in a high proportion of cases. When remissions were complete, S.M. and L.D.H. values returned to normal and remained so until relapse supervened. From these studies, we conclude that S.M. and L.D.H. are useful ancillary aids to the methods commonly employed for differentiating the types of leukemia, and that estimations of these enzymes also have some value in assessing the progress and prognosis of the disease.

Buoyant density and hybridization analysis of human DNA sequences, including three satellite DNAs

Total human DNA was fractionated from the three types of Cs2SO4 gradient used to prepare satellites I, II and III. Three satellite DNAs were found: satellite I with a mean buoyant density of 1.6888 g/ml comprising about 1.3% of the total, satellite II with a mean buoyant of 1.696 g/ml, comprising about 1% of the total an satellite III with a mean buoyant density of 1.699 g/ml comprising about 2.2% of the total. The buoyant densities of these satellites after purification were 1.686, 1.694 and 1.697 m/gl, respectively. A preparation with the attributes of satellite IV was isolated from the shoulder region of a satellite III preparative gradient. In situ hybridization using complementary RNA showed that the three satellites were located predominantly on chromosomes 9, Y, 15 and 1. Satellite II also showed marked hybridization to chromosome 16. Satellites I and II and III cross-hybridized to each other but satellites I and II did not. On the basis of our hybridization data, we suggest that some of the same sequences which comprise satellite III also comprise satellite I an II.

Metamorphosis of chronic granulocytic leukaemia arising in an extramedullary site

Abstract A 52 year old man with chronic granulocytic leukaemia, in whom splenectomy had been performed electively early in the disease, developed extramedullary leukaemic deposits in the stomach, in the skin and in lymph nodes. Marked chromosome abnormalities (hyperdiploidy and doubling of the Ph 1 chromosome) were found in a biopsied axillary lymph node at a time when marrow chromosomes showed only a 46XY Ph 1 positive pattern typical of the chronic phase. As metamorphosis of his disease progressed, cells with related chromosome abnormalities appeared in the blood. The sequence of events and cytogenetic changes suggested that cells responsible for metamorphosis appeared first in an extramedullary site and spread from there to the marrow and blood.

Differential synchronization of leukemic cells in vivo by hydroxyurea

Flow cytometry (FCM) was used to monitor the cell cycle changes which occurred in the bone marrow cells of a patient with acute myeloblastic leukemia following pre-induction treatment with hydroxyurea. Flow cytometric and cytogenetic analysis showed two populations of cells; one hypertetraploid and presumably leukemic, and the other diploid and possibly normal. Sequential FCM monitoring of bone marrow cells after hydroxyurea demonstrated an early rise (after 24 h) in the S-phase component of diploid cells and a rise in the S-phase component of hypertetraploid cells 48-72 h later. Conventional induction therapy timed to coincide with this latter peak resulted in early remission.

Satellite III DNA hybridised to chromosomes from patients with acute leukemia

Abstract Radioactive RNA complementary to normal human satellite III DNA was hybridised to previously Giemsa-banded metaphase chromosomes from six patients with acute leukemia. The distribution of satellite III DNA was found to be the same as in normal subjects, with hybridisation occurring in chromosome 9, the distal arm of the Y and to a lesser extent the centromeric region of chromosome 15. In two leukemic patients hybridisation confirmed that a trisomic or tetrasomic C-group chromosome was 8, as predicted by G-banding, and aneuploidy of chromosome 9 was not seen in any one case.

Haemopoietic inhibitors in aplastic anaemia—a review

&NA; Haemopoietic failure in aplastic anaemia is thought to be the result of a decreased flow of cells from the pluripotential stem cell (PSC) compartment into the morphologically identifiable proliferative compartments of erythropoiesis, granulopoiesis, monocytopoiesis and thrombopoiesis. In most cases there is probably a quantitative decrease in the size of the PSC reserve, in some the PSC might be qualitatively abnormal, while in a few the defect may reside not in the PSC itself but in the microenvironment in which it has to proliferate. In a significant proportion of patients with acquired aplastic anaemia PSC damage follows exposure to a drug or chemical, but in the remainder the aetiology is obscure.1 Although marrow transplantation offers promise for younger patients with suitable donors, treatment for the remainder will continue to be unsatisfactory until more is known of the cause or causes of PSC damage in ‘idiopathic’ aplastic anaemia. Until recently a major obstacle to progress has been our inability to study or quantitate haemopoietic stem cells in man. In the last decade however in vitro techniques have been developed for the study in human marrows of primitive granulopoietic progenitors, possibly the second‐generation stem cells committed to granulopoiesis (CFU‐C), and of primitive erythropoietic progenitors (or committed erythropoietic stem cells or CFU‐E). There have been few reports of successful techniques for studying human PSC in vitro, and most laboratories have concentrated on studies using CFU‐C and/or CFU‐E. Although interesting data have emerged, one needs constantly to keep in mind the fact that these analyses do not look at the PSC itself.

Granulocyte kinetic studies in the elucidation of clinical problems

Study of neutrophil granulocyte kinetics following the reinfusion of autologous DFP 32 -labelled granulocytes in man allows one to interpret the dynamic abnormalities of granulopoiesis in a wide variety of clinical disorders. The size of the total blood granulocyte pool (TBGP) can be calculated from the number of labelled granulocytes infused and the number found initially in the circulation, using isotope dilution principles. The TBGP is always greater than the circulating granulocyte pool (CGP), due to the presence of granulocytes marginated on the walls of vessels (the margined granulocyte pool, MGP). Granulocytes disappear from the blood in s random fashion, which can be characterized by the half disappearance time (T½) of labelled granulocytes in the blood. The granulocyte turnover rate (GTR) can be derived from the TBGP and the T½. In a steady-state situation the GTR is equal to the rate at which granulocytes are produced by the marrow. The authors used these techniques to study two groups of patients: (a) those with lymphoma, and (b) those with neutropenia due to non-malignant disease. Six of the 8 patients with lymphoma had splenamegaly at the time of study, and 4 of thesr were neutropenic. Five of the 7 other patients with neutropenia also had splenomegaly (three Feltys syndrome, one DLE, one portal hypertension). The majority of patients with splenomegaly had an increased MGP : CGP ratio, but none showed a significant shortening of granulocyte survival. In most cases, granulocytes were being produced at a normal or greater than normal rate. Two patients with splenomegaly (one with lymphoma, one with Feltys syndrome) underwent splenectomy, with an increase in circulating granulocytes in each case. Results in 2 patients with lymphoma without splenomegaly were essentially normal, while 2 patients with neutropenia without splenomegaly showed a normal granulocyte survival, a decrease in granulocyte production and an increase in the MGP CGP ratio.

Value of muramidase assay in clinical haematology

Muramidase (lysozyme) is a hydrolytic enzyme present in monocytes and all members of the granulocytic series from pro-rnyelocytes to neutrophil polymorphs. Measurable quantities of this enzyme are also present in serum, and in normal humans the level of serum muramidase correlates with the absolute granulocyte count. Studies performed by other investigators have related serum muramidase to leukaemia, and experimental work, in particular that performed by Fink and Finch, has demonstrated a correlation between serum muramidase and granulocyte turnover. In the Clinical Haematology Unit at Sydney Hospital the authors studied patients with both leukaemia and neutropenia of varying origin. Raised serum muramidase levels in acute myelomonocytic and some other myeloid leukaemias were confirmed. Among 27 patients with neutropenia studied to date, correlations have been made between the following parameters : serum and marrow muramidasc levels, clinical features, marrow and blood counts and morphology. A close correlation was demonstrated between levels of serum muramidase and marrow cellularity. All patients with a low serum muramidase had hypocellular marrow, while 14 of the 19 patients with a normal or elevated muramidase had a marrow which was either normo-or hypercellular. A highly significant correlation was also observed between serum muramidase levels and spleen size : all patients with splenomegaly had either normal or elevated serum muramidase levels irrespective of marrow cellularity. The ratio of marrow serum muramidase, normally 1.5-3 1, was also determined. The ratio was found to be elevated in 5 patients, 4 of whom showed clear morphological evidence of disturbed myeloid, proliferation. It was concluded that serum and marrow muramidase estimations may help in separating neutropenias due to decreased myelopoiesis from those caused by excessive granulocyte destruction.

In vitro studies of the granulopoiesis committed stem cell pool in haematologically normal subjects

It can be shown in experimental animals that there are two types of haemopoietic stem cells: (1) Totipotential stem cells which can give rise to all haemopoietic elements; (2) Stem cells committed respectively to erythropoiesis, granulopoiesis and thrombopoiesis. Totipotential stem cells can be assayed in the mouse, but not in man, by a spleen colony technique. Stem cells committed to granulopoiesis in the mouse can be assayed by an in-vitro method in which colonies are grown from marrow cells plated in soft agar tissue cultures. The number of these colonies in the mouse has been shown to measure the number of granulopoietic stem cells present. The authors and others have been able to grow similar colonies from human marrow in soft agar cultures. Growth of these colonies requires the presence of an appropriate stimulus which can be provided by intact peripheral blood leucocytes, or by medium incubated with peripheral blood leucocytes, or spleen cells in suspension culture. Stimuli effective for mouse marrow growth in agar (e.g. human urine or serum) are ineffective for the growth of human marrow. The colonies grown from human marrow have been shown to consist predominantly of granulopoietic cells and mature granulocytes, with occasional mononuclear cells. The growth characteristics of these cells were described. Studies of this type allow, for the first time, quantitative assessment of the size of one of the stem cell compartments in haematologically normal human bone marrow.