Normal sister chromatid exchange frequency in long-term survivors with acute leukemia.

We hypothesized that the sister chromatid exchanges assay in acute leukemia long-term survivors may detect: (a) long-term effects of combined chemo- and radiotherapy; and possibly (b) those individuals with inherently deficient DNA repair. Accordingly, we determined the sister chromatid exchanges frequency in 26 blood specimens from 24 acute leukemia long-term survivors (patients) and 14 blood specimens from 13 control subjects (controls). The patients consisted of 23 children with acute lymphocytic and one child with acute myelocytic leukemia. The median length of chemotherapy was 5 years. Eighteen of the 24 patients also received prophylactic fractional central nervous system irradiation for the first 3 years of treatment, and one patient received therapeutic irradiation to the central nervous system. The median off-therapy period at the time of study was 2.5 years with a range of 0 to 7.5 years. The controls consisted of the parents of the patients and laboratory personnel. A mean exchange score per cell was established for each specimen (25 to 30 cells/specimen were scored), and it ranged from 3.0 to 9.7 in the patients and from 3.0 to 11.5 in the controls. A mean +/- S.D. calculated from those means was 6.0 +/- 1.8 for the patients and 6.9 +/- 2.8 for the controls. They were not significantly different. We conclude that chemo- and radiotherapy produced no persistent DNA alterations detectable by this method.

Heterogeneity of human colony-forming cells.

Culture supernatants from established human fibroblast cell lines (FCM) and from phytahemagglutinin-stimulated peripheral blood leukocytes (LCM) were compared with respect to their stimulatory effects on the colony formation by human marrow leukocyte precursors in vitro using the methylcellulose culture system. The cultures were performed using specimens from children with a variety of disorders. LCM stimulation consistently produced a significantly higher proportion of macrophage colonies than did FCM stimulation, whereas FCM stimulation resulted in a significantly higher number of number of neutrophil colonies. Based on the sequential examination of colony numbers in 35 FCM-and 11 LCM-stimulated cultures, the colonies in LCM-stimulated cultures (predominantly macrophage colonies) survived much longer than those in FCM-stimulated cultures (predominantly neutrophil colonies). Cell fractionation experiments by velocity sedimentation on four different specimens revealed that the majority of FCM-responsive cells were large and formed pure neutrophil colonies, while the majority of LCM-responsive cells were smaller and formed either pure macrophage or mixed neutrophil-macrophage colonies. These observations suggest that human colony-forming cells consist of at least two distinct cell populations different in (1) cell size, (2) response to two different conditioned media, LCM and FCM, and (3) type of colonies each population forms.

In vitro microwave effects on human neutrophil precursor cells (CFU-C).

Human marrow cells were irradiated with 2450-MHz CW microwaves in a fluid-filled waveguide irradiation system. Cell exposure was conducted by placing a marrow cell suspension in 20-microliter glass microcapillary tubes were positioned in the exposure chamber, and irradiated at power densities from 31 to 1,000 mW/cm2 (with corresponding specific absorption rates of 62 to 2,000 mW/g) for 15 minutes. The temperature of the sample was maintained at a fixed point. Sham-irradiated (SI) and microwave-irradiated (MWI) cells were cultured in a methylcellulose culture system for neutrophil colony proliferation. There was no reduction in neutrophil colony number on days 6-7 or 12-14 in cells exposed at 31 or 62 mW/cm2, but as the power density was increased to 1,000 mW/cm2, there was a reduction in colony number of MWI cells compared with SI cells. The microwave interaction with the human neutrophil colony-forming cells was apparently not related to temperature rise, or to the state of cells cycle, and was irreversible.

Inhibition of bone marrow stem cell growth in vitro by methylmalonic acid: a mechanism for pancytopenia in a patient with methylmalonic acidemia.

A 7-week-old infant with methylmalonic acidemia had pancytopenia and hypoplastic bone marrow. The patient responded to large doses of vitamin B12 treatment, and within 3 wk, the blood counts and bone marrow cellularity returned to normal. To understand the mechanism of marrow depression in this infant, we examined the effect of the patient's plasma and methylmalonic acid itself on the in vitro growth of bone marrow-committed stem cells. The patient's plasma obtained before B12 treatment completely inhibited the marrow cell growth, whereas the posttreatment plasma showed no inhibition. Methylmalonic acid when added to the culture dishes in concentrations comparable to those reported in plasma of methylmalonic acidemia patients, inhibited growth of marrow stem cells in a concentration-dependent fashion. On the other hand, 16 to 18 hr incubation of cells in the same concentration of methylmalonic acid did not affect the recovery of viability of the cells. The observations suggest that methylmalonic acid is inhibitory to the proliferation of marrow stem cells. The mechanism of inhibition is yet to be elucidated.

Cytogenetics of juvenile type chronic granulocytic leukemia.

Cytogenetic examination of bone marrow cells from three patients with juvenile chronic granulocytic leukemia (CGL) showed 46,XX,3p-,11p+ ,t(11p:3p) in one case, 45XY,-E in another, and 45X(X),-C/47,XX,+G in the third. The case with the translocated chromosome originally presented like an acute lymphocytic leukemia (ALL). TAn overt clinical picture of juvenile CGL emerged two and a half years later. Serial study of this case revealed no cytogenetic abnormalities until two years after the diagnosis of ALL, when the translocated chromosome was first observed. Unlike the Ph1 chromosome in adult type CGL, chromosomal abnormalities in juvenile CGL lack specificity, resembling ALL in this respect.

Long-term cytogenetic studies in acute leukemia of children; the nature of relapse.

Sequential long-term cytogenetic studies in 71 children with acute leukemia were designed to investigate the nature of relapse after prolonged remission. In the overwhelming majority of the cases the findings suggested clonal identity of the leukemic cell population in relapse with that studied at the onset of the disease, notwithstanding considerable karyotypic instability in almost half of the patients. In a small minority an independent origin of the relapse clone could not be excluded on cytogenetic grounds but was considered unlikely, since mechanisms capable of accounting for the changes observed in these patients could be demonstrated in other cases. The persistence of diploid leukemic cells in the presence of an aneuploid subclone was demonstrated in the relapse bone marrow and/or spinal fluid in all active phases of the disease. On this basis the conversion from an aneuploid to a predominantly or exclusively diploid karyotype could be visualized, and a new model of clonal evolution, involving repetitive formation of abnormal karyotypes from a surviving diploid clone could be suggested.