Malignant leukemic cell separation by iron colloid immunomagnetic adsorption.

We describe an immunomagnetic assay applicable to bone marrow purging of leukemic patients before an autologous bone marrow transplantation. An iron colloidal suspension with a CD10 monoclonal antibody (MoAb) against the common acute lymphoblastic leukemia antigen (CALLA) covalently bound to the surface of the particles has been used. NALM-6 cells, a pre-B leukemic cell line expressing the CALLA, were labeled with supravital DNA dye (Hoechst 33342), mixed with peripheral blood, and incubated with the MoAb coated to iron particles. Using this reagent, at a cell concentration of 2000/microliters, a purging effect greater than 3.5 logs is observed with 0.1 mg of coated particles. Three successive rounds of treatment with the same coated particles at the same dose showed approximately the same depletion after each treatment. The recovery of clonogenic myeloid progenitors (granulocyte-macrophage colony-forming units; CFU-GM) is around 75%. No depletion was observed when the iron particles were not attached to the CD10 MoAb, or when they were attached to a MoAb not recognizing CD10+ cells. A comparison with another commercially available magnetic support was also performed in order to evaluate the performance of our assay, which appears simple, efficient, cheap, and capable of handling large volumes of cells in sterile conditions and minimal time.

Comparative effect of cisplatin, spiroplatin, carboplatin, iproplatin and JM40 in a human myeloid clonogenic assay.

The relative toxicities of cisplatin and its analogs, spiroplatin, carboplatin, iproplatin and JM40, were tested against normal human progenitor myeloid cells (CFU-GM) in a clonogenic assay. Cells obtained from five bone marrows were incubated for 60 min with various drug concentrations and plated. The mean inhibitory concentrations for 50% of the bone marrow colonies (IC50) were 15.6 micrograms/ml for cisplatin, 0.4 micrograms/ml for spiroplatin, 56.3 micrograms/ml for carboplatin, 36.3 micrograms/ml for iproplatin and 179.5 micrograms/ml for JM40. Ratios of the IC50s of the analogs with cisplatin as reference drug closely followed the corresponding ratios of the clinical maximum tolerated doses. This correlation between the CFU-GM assay results and the clinical myelotoxicity suggests that the assay is adequate for predicting myelotoxicity in vitro and selecting in vitro drug concentrations for the human tumor clonogenic assay.

In vitro chemosensitivity testing of flavone acetic acid (LM975; NSC 347512) and its diethylaminoethyl ester derivative (LM985; NSC 293015).

The antitumor effect of flavone acetic acid, LM975, and its diethylaminoethyl ester derivative, LM985, was studied in four human malignant cell lines [WiDr, a colon carcinoma; LICR (LON) HN-3, a tongue carcinoma; MCF7, a breast carcinoma; K-562, a leukemia] using a colorimetric assay based on the reduction of dimethylthiazol-2-yl-diphenyltetrazolium. The cell lines were exposed continuously for 4-6 days to drug concentrations ranging between 0.1 and 500 micrograms/ml. For LM975, the concentrations inhibiting the growth of the various cell lines by 50% were 200 +/- 10, 97 +/- 7, 171 +/- 16 and greater than 500 micrograms/ml for LICR (LON) HN-3, WiDr, MCF-7, and K-562, respectively. The corresponding concentrations for LM985 were 151 +/- 3, 36 +/- 4, 86 +/- 3 and 140 +/- 18 micrograms/ml, respectively. The difference between LM985 and LM975 was statistically significant for the WiDr and LICR (LON) HN-3 lines. We also evaluated the cytotoxic activity of the two agents on normal human marrow myeloid progenitor cells in a colony-forming assay. Continuous exposure to the drugs gave a dose-dependent inhibition. The concentrations inhibiting the growth by 50% were 76 +/- 31 micrograms/ml for LM975 and 134 +/- 41 micrograms/ml for LM985. One hour incubation with either compound had no toxic effect on the myeloid progenitor cells. In conclusion, LM975 and LM985 do not appear to have a specific cytotoxicity for tumor cells. Our results indicate that, in vitro, toxicity on bone marrow myeloid progenitor cells is concentration dependent. Considering the low plasma concentration found in man after i.v. administration of LM985, our observations correlate well with the absence of drug-induced myelosuppression in patients.

Comparison of chemotherapy with immunotherapy for maintenance of acute lymphoblastic leukemia in children and adults.

Two hundred and seventeen patients, 1-50 yr old, with acute lymphoblastic leukemia in complete remission were randomized to receive a 1-yr consolidation chemotherapy of either type P, comprising 7 different drugs, or type M, consisting of methotrexate interspersed with prednisone and vincristine. Thereafter, they were randomized a second time to receive a 4-yr maintenance of either chemotherapy or immunotherapy, comprised of allogeneic blasts and bacillus Calmette-Guérin (BCG). Consolidation P caused more toxicity than consolidation M. However, comparison between the consolidation therapies P and M showed no significant difference, neither for disease-free interval nor for duration of survival. Chemotherapy showed more lethal toxicity in adults than in children. Comparison between chemotherapy (C) and immunotherapy (I) as maintenance treatment showed a significant (p = 0.016) superiority of C for disease-free interval (DFI). The difference was even more pronounced (p = 0.009) in the group with less than 8 g/dl of hemoglobin (Hb) at diagnosis before therapy. On the other hand, for patients with more than 8 g/dl Hb at diagnosis, presumably those with T-ALL, no difference in DFI was seen. No difference has been seen so far between maintenance therapies I and C concerning the duration of survival. The patients who were receiving maintenance I when they relapsed and who were consequently retreated by chemotherapy, survived longer from relapse than those patients retreated for relapse while receiving maintenance C.

Increased levels of myeloid progenitor cells in the blood of patients with metastatic invasion of the marrow.

Granulocyte-macrophage clusters and colony-forming cells (CFU-C) in the peripheral blood have been studied in 26 cancer patients with neoplastic bone marrow involvement. The concentration of CFU-C in the blood of normal individuals and of cancer patients with no bone marrow invasion, ranged from 0 to 99 ml. In contrast, out of 27 cancer patients with marrow invasion, 9 (35%) showed a significant increase of blood CFU-C (100 to 21000/ml) and of those 5 (19%) showed an increase of blood colonies (41 to 9000/ml). There was a strong correlation between increased CFU-C or colony concentration and the presence of myeloid or/and erythroid immature cells in the peripheral blood. On the other hand, there was no apparent correlation between an increased CFU-C level and anaemia or abnormal blood leucocyte count or marrow fibrosis. These observations suggest that bone marrow involvement by neoplastic cells may cause spatial redistribution of the granulocyte macrophage progenitor cells.

Effect of 2 anti-arrhythmic drugs aprindine and moxaprindine on the replication capacity of murine and human haemopoietic cells.

Aprindine, a potent anti-arrhythmic agent, occasionally seems responsible for agranulocytosis. In order to study its potential haematological toxicity, 3 different in vitro tests were used: (a) the capacity of human and mice bone marrow to incorporate tritiated thymidine (3HTdR), (b) the capacity of stimulated human blood lymphocytes to incorporate 3HTdR and (c) the capacity of human granulocyte-macrophage stem cells to form colonies in agar. For all these tests aprindine was found to be toxic at concentrations close to the clinical therapeutic serum concentration. Moxaprindine, chemically very close to aprindine exhibits also an anti-arrhythmic activity. It was examined in the same tests in parallel with the study af aprindine. Moxaprindine also exhibited haematological toxicity in the tests but at a significantly higher concentration, approximately twice that of aprindine. Assuming that these in vitro tests are relevant to the in vivo haematological toxicity, moxaprindine could be considered a clinically safer anti-arrhythmic agent than aprindine.

A case of chronic aneosinocytosis.

A 59-year-old woman, after complete recovery from an episode of drug-induced agranulocytosis, was found to sustain a chronic absence of recognizable mature and immature eosinophils in blood and bone marrow during a follow-up period of 8 years. Her bone marrow and peripheral blood cells cultured in vitro were able to produce normal numbers of eosinophil colonies. The present disorder was thus not caused by a lack of EO-CFU-C, nor by a defect in EO-CSA, nor by an immunologically mediated mechanism acting on mature or immature eosinophils or on EO-CSA producing cells. It is suggested that the marrow was not able to differentiate normally along the eosinophil pathway, possibly owing to a microenvironmental defect or, less likely, an intrinsic stem cell defect.

Treatment of brain giomas with high dose of CCNU and autologous bone marrow transplantation.

Seven patients with recurrent brain gliomas were treated by a single dose of CCNU 390 mg per m2. In five cases, chemotherapy was followed by autologous bone marrow transfusion containing 1.5 to 3 X 10(8) nucleated cells, 2.8 to 18 X 10(4) clusters plus colonies and 0.4 to 5 X 10(4) colonies forming cells per kg of body weight. Two patients were not grafted. None of these patients showed a clear cut response to the treatment as judged by clinical improvement and changes of the brain CT-scan. In 3 patients blood toxicity occurred early and was severe. In 4 others, it was milder and delayed. The duration and the severity of blood toxicity were modified by bone marrow transfusion but only slightly.

The leucocyte alkaline phosphatase activity in mature neutrophils of different ages.

The marrow myeloid precursor cells of a haematologically normal patient were labelled by intravenous injection of tritiated thymidine. Young labelled segmented neutrophils were then released from the marrow into the blood by an injection of cortisol. These PMN had a significantly lower LAP activity than the older blood neutrophils. It is shown that within the morphologic boundaries of the segmented PMN, the cells are still in different stages of cytoplasmic maturation. In addition, labelled and unlabelled neutrophils showed a linear and parallel increase of LAP activity during the 24 h following cortisol injection. This observation suggests that the neutrophil prematurely released in the blood can mature into normal LAP=PMN and more generally that LAP activity of a blood neutrophil increases with time.

Treatment of chronic myeloid leukemia.

Most known chemotherapy agents, used singly, may induce remission of CML. Those that best alleviate symptoms (anemia, splenomegaly, etc) do not change either the Ph' chromosome anomaly in the hemopoietic cells nor the median delay of 3--4 years before fatal blastic transformation. A new goal has recently been formulated in CML therapy, namely complete remission, i.e. disappearance of all signs of leukemia including the Ph' abnormality. This can be achieved in certain cases by the use of aggressive (radio)chemotherapy during the chronic phase of CML. Whether or not it means cure of CML awaits further investigation. Encouraging results with these new therapeutic modalities during the chronic phase, and the possibility of rescuing toxic marrow aplasia by autologous cryopreserved blood stem cells, make it less and less defensible to await CML transformation before using aggressive "eradication" therapy.

Clonal origin of a T cell lymphoproliferative malignancy.

A woman with a T cell lymphoproliferative malignacy and heterozhgosity at the X chromosome-linked locus for glucose-6-phosphate dehydrogenase (G-6PD) isoenzymes was studied to find the clonal origin of her circulating neoplastic T cells. The red blood cells, polymorphonuclear cells, whole mononuclear cells, and T cell-depleted mononuclear cells contained both A and B isoenzymes of G-6-PD. In contrast, the tumor cells, separated by using their capacity to form rosettes with sheep red blood cells, contained only the B isoenzyme of G-6-PD. This observation strongly suggests the monoclonality of this T cell malignancy.

Cell kinetics in chronic lymphocytic leukaemia (CLL).

The fractional production rate of CLL lymphocytes, certainly in the peripheral blood and possibly in the lymph nodes, is often lower than normal. On the other hand, the absolute production in CLL blood and in lymph nodes seems to be considerably increased in most cases. Arguments for an increased lymphocyte life span in CLL, although fragmentary and dispersed, exist. The blood CLL lymphocytes, as normal lymphocytes, exchange rapidly with an extravascular pool of lymphocytes. However, recirculation of lymphocytes--from blood to blood via the lymph node and the thoracic duct--does not proceed normally in CLL. Whether this is related to the B nature of the CLL lymphocyte, to the leukaemic nature of the CLL lymphocyte or to mechanical factors--crowding of the extravascular pool--is still debatable.

Hairy cell leukemia: functional, immunologic, kinetic, and ultrastructural characterization.

A diagnosis of hairy cell leukemia was made by optic microscopy, phase-contrast microscopy, electron microscopy, scanning microscopy, and histochemistry of the abnormal blood cells. In vivo these cells were found to have a half-time in the blood of approximately 150 hr. In vitro they had the capacity to adhere firmly to plastic, making it possible to obtain a pure population of hairy cells. Neither T-rosette formation nor phytohemagglutinin (PHA) transformation could be demonstrated in these cells. On the other hand, the presence of immunoglobulins on the surface of the hairy cells (HC) by immunofluorescence, and the synthesis and secretion by these cells of IgM type lambda-chains shown by radioimmunodiffusion, were in favor of their B-type lymphocyte origin. Similarities to chronic lymphocytic leukemia were apparent.