Screening of cytotoxic drugs for residual bone marrow damage.

Several cytotoxic drugs were tested for their ability to produce permanent residual damage to the bone marrow. A short course of the drug was given to BALB/c female mice, and the numbers of various types of bone marrow cells were determined at least two months later. Evidence of residual damage was found after administration of busulfan and 1,3-bis(chloroethyl)-1-nitrosourea, but not after administration of cyclophosphamide, 5-fluorouracil, 6-mercaptopurine, methotrexate, or vinblastine.

Increased mutation frequency following treatment with cancer chemotherapy.

The relationship between somatic mutation and cancer was studied by measuring in vivo mutation frequency and in vitro mutability using lymphocytes from 28 untreated adult patients with solid tumors, 14 untreated patients with lymphoma, and 27 patients with solid tumors or lymphoma who had been treated with chemotherapy and/or radiotherapy. In vivo mutation frequency in untreated patients did not differ from that of controls, except perhaps in patients with lymphoma, who showed a slight increase. Lymphocytes from untreated patients with solid tumors or lymphoma did not show a greater increase in mutations induced after X-radiation or UV radiation than did lymphocytes from controls. For all the untreated patients, the geometric mean mutation frequency was 6.72 X 10(-6), and it was significantly increased to 19.57 X 10(-6) following chemotherapy and 34.40 X 10(-6) following chemotherapy and radiotherapy. The results suggest that excessive systemic exposure to mutagens or inherent susceptibility to mutagenesis are not important etiological factors in at least the majority of patients with cancer. The mutations produced by treatment may be related to the late side effects of therapy such as second neoplasms.

Mutation rate of normal and malignant human lymphocytes.

The genetic stability of normal and neoplastic lymphocytes was compared by using base-line mutation frequency and mutation rate/cell generation. Mutations at the hypoxanthine-guanine phosphoribosyltransferase locus were studied by enumerating thioguanine-resistant cells in a clonogenic assay. The base-line ("spontaneous") mutation frequency was 1.52 X 10(-6), 6.38 X 10(-6), and 1.06 X 10(-6) for normal cells from three individuals and was 1.16 X 10(-3), 6.08 X 10(-5), and 3.06 X 10(-5) for the three malignant cell lines, Jurkat (JM), HRIK, FMC-Hu1B, respectively. The mutation cell/generation rate was 24.6 X 10(-8), 15 X 10(-8), and 5.5 X 10(-8) for lymphocytes from the three normal individuals, and 666.4 X 10(-8), 52.8 X 10(-8), and 131 X 10(-8) for the three malignant cell lines. The results suggest that neoplastic lymphocytes are more genetically unstable than normal lymphocytes.

Molecular nature of in vivo mutations in human cells at the autosomal HLA-A locus.

The mutations present in vivo in normal human cells were studied at the HLA-A locus by isolating mutant lymphocytes using antibody-complement immunoselection and cloning at limiting dilution. The molecular basis for mutation in 127 mutant lymphocytes from 10 individuals was determined by studying a variety of polymorphic gene loci on both arms of chromosome 6. No change was detected in 78 mutants (61.4%), gene deletion was detected in 11 (8.7%), and mitotic recombination was detected in 38 (29.9%). Neither gene conversion nor chromosome loss was detected. These observations document the mechanisms responsible for gene loss in normal human cells in vivo, emphasize the importance of mitotic recombination, and indicate the similarity between mutational mechanisms in normal cells and in cancer cells.

Early resistance to therapy during induction in childhood acute lymphoblastic leukemia.

Many patients with acute lymphoblastic leukemia (ALL) are not cured by current therapy because of the development of drug resistance. It is not clear when resistance develops during the growth of the leukemic clone and whether resistant cells are already present at diagnosis or develop later during treatment. Twenty-two uniformly treated children with ALL were studied throughout induction treatment. The size of the leukemic clone in blood and marrow was estimated by limiting dilution PCR analysis, using the rearranged immunoglobulin heavy chain gene as a molecular marker. The decline in the number of leukemic cells was biphasic in virtually all patients. For both marrow and blood, the logarithmic mean of the number of leukemic cells fell by approximately four orders of magnitude during the first 2 weeks, one order of magnitude during the third week, and not at all during the last two weeks of induction treatment. For marrow, the median of the fraction of leukemic cells in each patient that survived per week of treatment was 0.008 for the first 2 weeks, 0.12 for the third week, and 1.4 for the last 2 weeks; for blood, the corresponding figures were 0.003, 0.14, and 0.69, respectively. In individual patients, the results for marrow and blood showed good correlation. The biphasic decline of leukemic cell number suggests that most leukemic cells were sensitive to treatment and were rapidly killed, leaving behind a minor but substantial population of drug-resistant cells. The most likely explanation for this phenomenon is that these resistant cells were already present at diagnosis, their resistance having originated from genetic or epigenetic mutations during prior growth of the leukemic clone.

Molecular diagnosis of Philadelphia negative CML using the polymerase chain reaction and DNA analysis: clinical features and course of M-bcr negative and M-bcr positive CML.

The Philadelphia chromosome (Ph) is the cytogenetic hallmark of chronic myeloid leukemia (CML) and as such has been used to confirm the diagnosis of CML based on morphological and clinical criteria. We have investigated 12 patients who were considered to have clinical and morphological features of CML and who did not have detectable abnormalities of chromosomes 9q34 or 22q11. In six of the 12 patients, rearrangement within the 5.8 kb major breakpoint region (M-bcr) and amplification of CML specific M-bcr-ABL cDNA sequences by the polymerase chain reaction (PCR) was demonstrated. Six other CML patients did not have rearrangement of the M-bcr gene or amplification of BCR-ABL by PCR. These patients had atypical CML. They were significantly older, most had less than 10% immature granulocytic cells (metamyelocytes, myelocytes and promyelocytes) and had various degrees of marrow fibrosis. Three of these six patients died of blastic transformation at 4, 15 and 54 months from diagnosis.

The incidence and prognostic significance of mutations in codon 13 of the N-ras gene in acute myeloid leukemia.

To determine the incidence and prognostic significance of mutation in the N-ras gene in de novo acute myeloid leukemia (AML) we performed an analysis of bone marrow smears from 219 patients with de novo AML treated between 1984 and 1986 and followed for at least six years. DNA extracted from bone marrow smears taken at diagnosis was screened for the presence of mutations in codons 12 and 13 of exon 1 by using the polymerase chain reaction to insert an Hph1 restriction enzyme site into DNA. Presumptive mutations were confirmed by direct sequencing. Mutations were detected in a total of 26 patients (12%); in nine patients (4%) in codon 12 only, in ten patients (5%) in codon 13 only, and in seven patients (3%) in both codons. Mutations in codon 12 or codon 13 were not associated with any clinical features. Mutations in codon 12 had no prognostic significance but mutations in codon 13 were associated with an increased remission rate, a more durable remission, and a significantly prolonged survival which appeared to be independent of other prognostic factors.

Characterization of clonal immunoglobulin heavy chain and I cell receptor gamma gene rearrangements during progression of childhood acute lymphoblastic leukemia.

Instability of antigen receptor gene rearrangements during progression of acute lymphoblastic leukemia (ALL) has important implications for polymerase chain reaction (PCR)-based techniques using these genes for the detection of minimal residual disease (MRD). Antigen receptor gene instability may lead to false negative results in bone marrow samples taken during remission. Utilizing the PCR and consensus primers for rearranged immunoglobulin heavy chain (IgH) and T cell receptor gamma (TCR gamma) gene sequences, we analyzed the bone marrow samples at diagnosis and first relapse for 37 children with ALL. The incidence of clonal evolution at the IgH locus was 9/33 (27%) and at the TCR gamma locus 1/15 (7%). In four of the nine patients with clonal evolution at the IgH locus, the sequence at relapse retained the diversity and joining region (D-N-J) sequences from diagnosis. Patients with clonal evolution were characterized by a higher incidence of more than one IgH PCR band at diagnosis and by late relapse (> 18 months from diagnosis). These results suggest that, where possible, patients with more than one IgH PCR rearrangement at diagnosis should be monitored using another antigen receptor gene, such as TCR gamma, since evolution for this gene was found to be a rare event. By combining this approach with a strategy directed at the more stable D-N-J region of the IgH gene, MRD false negativity would have occurred in less than 10% of patients in the present study.

The use of monoclonal gene rearrangement for detection of minimal residual disease in acute lymphoblastic leukemia of childhood.

Sensitive quantification of minimal residual disease (MRD) using the polymerase chain reaction (PCR) is strongly predictive of outcome in childhood acute lymphoblastic leukemia (ALL), with MRD levels at the end of induction therapy of >10(-3) predicting a poor outcome. Methods for sensitive quantification are, however, complicated and time-consuming. Detection by PCR of monoclonal immunoglobulin heavy chain (IgH) and T cell receptor (TCR) gene rearrangements is simple and can be used in routine laboratories but is non-quantitative and of lower but uncertain sensitivity. The aim of this study was to determine the value of detection of monoclonality in identification of different levels of MRD. We looked for monoclonality in 64 bone marrow aspirates which had been obtained from 31 patients with B lineage ALL at various times during induction therapy and for which levels of MRD had been determined by limiting dilution analysis using patient-specific PCR primers. Detection of monoclonality identified levels of MRD of > or =10(-3) during induction with a sensitivity of 78% and a specificity of 93%. The positive and negative predictive values were 0.86 and 0.88, respectively. The sensitivity of detection of a monoclonal IgH rearrangement was greater than that for the TCRgamma locus during induction as an IgH rearrangement was detected more often than a TCRgamma rearrangement in patients who had both IgH and TCRgamma rearrangement at diagnosis. Detection of monoclonality is therefore a simple and quick test applicable to the majority of patients with ALL and it may be useful in identifying high-risk patients at the end of induction and in identifying relapsing patients later during therapy.

Comparison of methods for assessment of minimal residual disease in childhood B-lineage acute lymphoblastic leukemia.

The level of minimal residual disease (MRD) early in treatment of acute lymphoblastic leukemia (ALL) strongly predicts the risk of marrow relapse. As a variety of methods of varying complexity have been separately used for detecting and quantifying MRD, we compared the prognostic utility of three methods measurement of blast percentage on day 14 of treatment, detection of monoclonality on day 14 or day 35, and measurement of MRD by PCR-based limiting dilution analysis on day 14 or day 35. The study group comprised 38 children aged 1-15 with Philadelphia-negative B-lineage ALL who were uniformly treated and followed until relapse or for a minimum of 5 years. We also studied some of the technical factors which influence the ability to detect MRD. Measurement of blast percentage on day 14 by an expert morphologist, detection of monoclonality on day 35, and PCR-based measurement of MRD levels on days 14 and 35 all showed significant ability to divide patients into prognostic groups. Measurement of blast percentage on day 14 by routine morphology or detection of monoclonality on day 14 were not useful. The quality of DNA samples varied greatly, as determined by amplifiability in the PCR. However, virtually all amplifiable leukemic targets in a sample were detectable which suggests that the level of detection achieved by limiting dilution analysis is essentially determined by the amount of DNA which it is practicable to study. We conclude that quantification of MRD at the end of induction provides the full range of prognostic information for marrow relapse but is complex; detection of monoclonality on day 35 is simple and has good positive predictive value; and quantification of MRD on day 14 merits further study. PCR-based methods for measurement of MRD levels should incorporate a correction for variation in DNA amplifiability.

Effect of the Philadelphia chromosome on minimal residual disease in acute lymphoblastic leukemia.

The Philadelphia translocation is associated with a poor prognosis in adults and children with acute lymphoblastic leukemia, even though the majority of patients achieve remission. To test the hypothesis that the translocation leads to drug resistance in vivo, we studied 61 children and 20 adults with acute lymphoblastic leukemia and used the level of minimal residual disease at the end of induction as the measure of drug resistance in vivo. In children the presence of the translocation was associated with a significant increase in residual disease, indicating higher drug resistance in vivo; five of seven Philadelphia-positive children but only five of 54 Philadelphia-negative children had a minimal residual disease level >10(-3), a level which is associated with a high risk of relapse in childhood acute lymphoblastic leukemia of standard risk. By contrast, in adults, residual disease and hence drug resistance was already higher than in children, and the presence of the Philadelphia translocation in seven patients had no obvious additional effect. We conclude that the Philadelphia chromosome may increase resistance to drugs in vivo in children, but not detectably in adults.

The mononuclear phagocyte system in experimental chronic marrow failure.

An animal model of chronic hypoplastic marrow failure (CHMF, aplastic anaemia), produced in mice by busulphan, was studied for changes in function and number of tissue macrophage populations. In vitro study of peritoneal macrophages showed no abnormality of function as assessed by chemotaxis, spreading, phagocytosis or bacterial killing. In vivo study of Kupffer cells showed a normal total number and a normal rate of clearance of intravenously injected colloidal carbon. Kupffer cells showed a reduced turnover rate as assessed by a cytoplasmic double-labelling technique and a reduced rate of carbon clearance following stimulation with endotoxin. The numbers of splenic and peritoneal macrophages were both to be decreased and the peritoneal macrophages also showed a decreased recruitment in response to intraperitoneal injection of casein. This decreased recruitment was corrected following marrow transplantation. These observations suggest that in experimental CHMF different tissue macrophage populations are variably decreased in number and that the recruitment of new macrophages is decreased, particularly in response to stimulation. The observations on peritoneal macrophages suggest that at least some of the changes can be directly related to the lesion of marrow precursors.

The cells of origin of lymphocyte clones grown by limiting dilution.

A high proportion of the mononuclear cells separated from peripheral blood by Ficoll-Hypaque centrifugation show extensive proliferation when cloned at limiting dilution. The nature of clone-forming cells ( CFCs ) and the cells generated during clone formation were studied by cytotoxicity with antibody and complement and by immunofluorescence. Cytotoxicity prior to cloning with OKT3 plus OKT11 eliminated 99.34% of clones, indicating that virtually all CFCs are T-lymphocytes, and cytotoxicity with OKT4 or OKT8 indicated that helper and suppressor subclasses each contribute approximately half of the CFCs . Immunofluorescence of cells proliferating in clones confirmed that all clones were T-lymphocytes and showed that 47% were OKT4 positive and 47% were OKT8 positive; 6% were negative with both OKT4 and OKT8. The results indicate that in the nearly optimal conditions for proliferation provided by limiting dilution, clones arise entirely from T-lymphocytes and clone formation is a property of a variety of lymphocyte subclasses.

An improved method for the growth of human lymphocyte colonies.

An improved method has been developed for growth of human lymphocyte colonies in agar. The method is a single-step assay involving the addition of irradiated lymphocytes which do not themselves form colonies but which stimulate a small number of non-irradiated cells to do so. Over the range 0 to 1 x 10(5) target lymphocytes, there is a linear relationship between number of lymphocytes cultured and number of colonies produced. Technical factors which influence colony growth include osmolality of the medium, foetal calf serum concentration, agar concentration, PHA type and concentration, and 2-mercaptoethanol. Colony formation is not affected by using homologous irradiated cells from an individual of the same age. For normal individuals aged 11 to 45, colonies of 20-200 cells were grown to a plating efficiency of 2.18%, with a range 1.28-4.00%. The advantages of the method are its simplicity, the linear relationship between cells plated and colonies formed, which enables the method to be used as a quantitative assay, and the ability to determine whether an observed abnormality is due to an abnormality of colony forming cells or of irradiated stimulating cells.

Purging of myeloma cells using all-trans retinoic acid in a mouse model.

OBJECTIVE: The 5T33 murine model of multiple myeloma was used to investigate the potential of all-trans retinoic acid (ATRA) to purge clonogenic myeloma cells from autologous hemopoietic stem-cell harvests by differentiating immature 5T33 cells into terminal-stage plasma cells with limited repopulation capacity. MATERIALS AND METHODS: 5T33 cells were treated with 10 microM ATRA and the effect on cell clonogenicity was determined by measuring the time to paraprotein detection in C57Bl/KaLwRij mice compared to control animals. Cell differentiation and apoptosis following ATRA treatment were investigated using flow cytometry and caspase-3 assay. Treatment with ATRA resulted in a 33% reduction in the in vitro cloning efficiency of 5T33 cells. Reduced in vitro clonogenicity of 5T33 cells following ATRA treatment was supported by a 16-49% increase in the time taken for C57Bl/KaLwRij mice to develop paraprotein following injection of 5T33 cells pretreated with ATRA for 8 days. Although ATRA was shown not to alter the in vitro growth characteristics of 5T33 cells, significant inhibition of apoptosis was observed. RESULTS: Treatment with ATRA also resulted in an increase in the proportion of 5T33 cells expressing the CD54 adhesion molecule, which is known to be highly expressed on mature myeloma cells. CONCLUSION: The ability of ATRA to decrease the clonogenicity of 5T33 cells in vitro and increase the time to disease development in vivo suggests that this drug may be useful for purging autologous stem cell harvests in the clinical setting.

A proliferative defect of marrow cells in experimental chronic hypoplastic marrow failure (aplastic anaemia).

Marrow cells from control mice and mice with chronic hypoplastic marrow failure (CHMF, aplastic anaemia) were grown in tissue culture and growth was assessed by measuring the number of cells/colony and uptake of 3H-thymidine/colony. Cells from mice with CHMF showed impaired proliferation in response to colony stimulating factor. Mixing experiments suggested that the impairment of proliferation was not dut to alteration in suppressor or helper cells but to an intrinsic lesion of the marrow cells themselves.

Cloning of human lymphocytes using limiting dilution.

A simple technique for cloning lymphocytes at limiting dilution in microwells has been developed using phytohaemagglutinin, irradiated cells and conditioned medium containing interleukin-2. For freshly isolated peripheral blood lymphocytes, the mean frequency of clone forming cells (CFC) was 29.4% and for continuously cultured lymphocytes it was 25.5%. Quantitative considerations, mixing experiments involving HLA-A2 positive and negative cells and replating experiments indicated that growth in most of the positive microwells originated from a single cell which had a very high self-renewal capacity and underwent approximately 15 divisions during the 14 days of culture. The high plating efficiency and sustained growth observed with this technique suggests that it is the method of choice for enumerating clone forming cells or for isolating clones.

Ageing in vivo does not influence micronucleus induction in human lymphocytes by X-irradiation.

To test the hypothesis that the age-related decline in the stability of the genome is the consequence of an increasing deficiency in DNA repair we compared the extent of chromosome damage, after X-irradiation, in lymphocytes from healthy young and old individuals, using the expression of micronuclei as the end-point. Micronuclei have been shown to increase in number with age and they were enumerated using a recently described and improved technique which involves measurement of micronuclei in cells that were blocked from performing cytokinesis. The level of X-ray-induced micronuclei in cytokinesis-blocked cells, after exposure to 75 cGy and 150 cGy, was measured by subtracting the base-line micronucleus frequency in the control unirradiated cultures from the observed micronucleus frequency in the irradiated cultures. There was no difference between the results for the young and old subjects thus indicating that cells from the aged subjects do not exhibit increased chromosomal instability following X-irradiation. These results suggest that repair of those DNA lesions that lead to chromosome breakage does not decline with age.

Human lymphocytes resistant to 6-thioguanine increase with age.

Using an autoradiographic technique we determined the number of circulating lymphocytes that were resistant to 6-thioguanine and which were presumably mutants at the hypoxanthine--guanine phosphoribosyl transferase locus. The number in normal individuals was found to increase exponentially with age. The data suggest a relationship between mutagenesis and ageing, perhaps by way of a decline with age in the fidelity of DNA replication or repair.