Resistance circumvention strategies tested in clinical leukaemia specimens using the MTT colorimetric assay.

We have used a 4-day MTT colorimetric assay to study drug sensitivity of leucocytes from leukaemia patients and from normal donors. Response to Adriamycin, vincristine, aclacinomycin A, 3'-deamino-3'-morpholino-13-deoxo-10-hydroxycarminomycin (MX2), and melphalan has been determined, together with the effects of the resistance modifiers verapamil, cyclosporin A, and ethacrynic acid. Sensitivity of chronic lymphoblastic leukemia (CLL) lymphocytes to vincristine was much greater than that of normal lymphocytes or of leucocytes from myeloid leukaemia patients. These cells were also more sensitive to melphalan. Verapamil and cyclosporin A at clinically achievable doses of 1 microgram/ml produced significant chemosensitisation in normal and leukaemic specimens, but the sensitisation ratio was greater than or equal to 2 only in a minority of specimens, except in the case of sensitisation to vincristine seen in the majority of CLL specimens. Sensitisation was generally greater in the more chemo-resistant specimens. The ratio of sensitivities of cells to Adriamycin compared with aclacinomycin A was greatest in the more Adriamycin-resistant specimens which supports the idea that cross-resistance between these agents may not be great. This was not, however, true for the ratio of Adriamycin/MX2 sensitivity. Use of the MTT assay may allow the identification of patients who would benefit from treatment with resistance modifiers or with 'low-resistance' anthracyclines.

Selective toxicity of ethacrynic acid towards lymphocytes of chronic lymphocytic leukemia in vitro.

We have used the MTT colorimetric assay to determine the sensitivity to ethacrynic acid of lymphocytes from normal donors and of peripheral blood cells from leukaemia patients. Whereas normal lymphocytes and cells from acute or chronic myeloid leukaemia showed similar sensitivities (median inhibitory dose, ID50 = 20-22 microM), lymphocytes from chronic lymphocytic leukaemia patients were much more sensitive (ID50 = 6 microM). This result was found irrespective of the assay duration.

Chemotherapy versus transplants for acute myelogenous leukemia in second remission.

The best therapy for persons with acute myelogenous leukemia (AML) in 2nd remission is unknown. Bone marrow transplants from an HLA-identical sibling are reported to be better than chemotherapy but this is controversial. The objective of the study was to compare 3-year leukemia-free survival (LFS) in comparable subjects receiving chemotherapy or a transplant. 485 persons with AML in 2nd remission were studied. The chemotherapy cohort included 244 persons treated on trials of the British Medical Research Council, Eastern Cooperative Oncology Group and MD Anderson Hospital. The transplant cohort included 257 persons transplanted worldwide and reported to the international Bone Marrow Transplant Registry (16 were also chemotherapy subjects.) Subjects were selected for comparable age and year of treatment. Preliminary analyses identified two factors correlated with LFS: age < or = or > 30 years and 1st remission duration < or = or > 1 year; subsequent analyses were partitioned accordingly. Three-year probabilities of treatment-related mortality with chemotherapy and transplants were 7% (95% confidence interval, 3-15%) vs 56% (49-63%). Three-year leukemia relapse probabilities were 81% (74-86%) vs 41% (33-49%). Three-year probabilities of LFS were 17% (12-23%) vs 26 (20-32%). Cohort analysis showed significantly higher LFS with transplants vs chemotherapy in persons < or = 30 years and 1st remissions > 1 year (41% (29-53%) vs 17% (7-32%); P = 0.017) and those in > 30 years with 1st remissions < or = 1 year (18% (9-29%) vs 7% (2-16%); P = 0.046). Others had comparable LFS with both treatments. These data indicate better LFS with HLA-identical sibling transplants than chemotherapy in some persons with AML in 2nd remission.

Adverse impact of bone marrow transplantation on quality of life in acute myeloid leukaemia patients; analysis of the UK Medical Research Council AML 10 Trial.

The increasing success of intensive consolidation chemotherapy (CCT) as an alternative to bone marrow transplant (BMT) in acute myeloid leukaemia (AML) necessitates comparison of the impact on quality of life (QoL) of these two treatment modalities. Most QoL studies following BMT involve small patient numbers and provide ambivalent results. The present study examines QoL in a large number of patients 1 year from the end of treatment within the United Kingdom Medical Research Council (UK MRC) AML10 trial of BMT versus CCT. Allogeneic-BMT (Allo-BMT) was observed to have an adverse impact on most QoL dimensions compared with Autologous-BMT (A-BMT) and CCT. More patients receiving BMT had mouth dryness problems and worse sexual and social relationships, professional and leisure activities than CCT patients. QoL in A-BMT patients was less impacted than Allo-BMT. Intention-to-treat analysis showed similar results. These results indicate that a reconsideration of treatment strategies is warranted, and that further, good prospective studies are needed to evaluate more clearly the effects of these treatments in long-term survivors.

Comparison of 1 + 5 DAT and 3 + 10 DAT followed by COAP or MAZE consolidation therapy in the treatment of acute myeloid leukemia: MRC ninth AML trial.

This trial, which will probably accrue over 1,000 patients before it closes, has already demonstrated that high remission rates are attainable in large multicenter studies using 'conventional' doses of ara-C. The results in the group aged less than 40 are comparable with many single-center studies. There is, at the moment, only a borderline difference in the remission rates between the two forms of induction therapy but the more intensive regimen of DAT 3 + 10 achieves remission more quickly and requires less supportive care. Analysis of the reasons for failure to enter remission continues to show that inadequate supportive care remains an important reason why the remission rates are not higher. Drug-related deaths in remission, though decreasing gradually in this study, are a disturbing consequence of increasing the intensity of consolidation treatment.

Effect of polar-planar compounds on the differentiation of normal human myeloid immature marrow cells.

Polar-planar compounds which are potent differentiating agents, have been reported to enhance the response of friend erythroleukaemic cells and other malignant cell lines to mitomycin C, cis-platinum and radiation. With the upcoming of phase II clinical trials of these polar compounds alone and in combination with cytotoxic agents, the effect of 3 polar compounds: dimethylsulphoxide, dimethylformamide and hexamethylene bisacetamide on the differentiation of normal human myeloid immature marrow cells was studied. The results showed that a long-term exposure to these polar compounds may lead to a premature senescence of these marrow progenitors.

Translocation 5;21 and interstitial deletion of chromosome 7 in a case of chronic myelomonocytic leukemia.

We report a case of chronic myelomonocytic leukemia with a translocation involving chromosome 5 and 21, namely, t(5;21)(q35.3;q22.1), and an interstitial deletion of the long arm of chromosome 7. High-resolution analysis at the 900-band stage has shown that the lesion in chromosome 7 is an interstitial deletion involving loss of the segments q22-q35 of the long arm of chromosome 7 and retaining the telomere.

T-Cell Receptor and Immunoglobulin Gene Rearrangements in Acute Myeloid and Undifferentiated Leukemias of Adults: Correlation with Weak Surface Expression of CD45 and CDw52 Antigens.

We examined 150 cases of acute myeloid leukemia (AML) and 8 cases of acute undifferentiated leukemi (AUL) of adults for both phenotypic and genotypic evidence of commitment to the lymphoid lineages. There was no correlation between expression of lymphoid differentiation antigens and rearrangement of T-cell receptor (TCR) and immunoglobulin (Ig) DNA sequences. In particular 8 cases of CD7(+) AML were germline for all TCR α, ß,µ, and δ Also none of the 11 cases with IgJH rearrangement expressed CD19 either on the cell surface or within the cytoplasm. Ten out of 13 cases with TCR/Ig gene rearrangement were either cytologically undifferentiated AML of FAB type M1 or AUL. None had clear immunophenotypic evidence for commitment to lymphoid lineages but 9 expressed only very small amounts of CD45 'framework' antigens and 7 expressed small amounts of CDw52 (CAMPATH-1). This phenotypic combination is otherwise seen only in precursor B cell ALL. TCR/Ig gene rearrangement in AML and AUL of adults appears to have become dissociated from the rest of the lymphoid differentiation 'program'. Recognition of these cases may be facilitated by the weak expression of both CD45 and CDw52 antigens.

Triple combination of retinoic acid+aclacinomycin A+ dimethylformamide induces differentiation of human acute myeloid leukaemic blasts in primary culture.

Differentiation induction therapy provides unalternative for treatment of acute myeloid leukaemia (AML) patients who are either unsuitable for or unresponsive to conventional cytotoxic chemotherapy. The effect of a triple combination of retinoic acid(RA)+aclacinomycin A (ACM)+dimethylformamide (DMF) on differentiation of blasts from 24 AML patients was studied. Non-adherent mononuclear cells were seeded at a concentration of 5 x 10(5) cells/ml in 24-well tissue culture plates containing RPM11640 culture medium with 20% fetal calf serum and 10% 5637-conditioned medium and incubated with 10(-6) M retinoic acid, 80nM aclacinomycin A and/or 100mM dimethylformamide alone and in combinations with each other for six days at 37 degrees C in a humidified incubator under 5% CO2. Morphological, cytochemical and functional differentiation into mature cells were induced in blasts from 22 out of the 24 AML patients after exposure to the triple combination of 10(-6)M RA+80nM ACM+100mM DMF for 6 days in primary culture. These highly effective results justify a clinical trial of this triple combination for AML patients who are either unsuitable for or unresponsive to conventional cytotoxic chemotherapy.

Auer bodies in acute myeloid leukaemia patients.

Auer bodies have been reported in several clinical studies to be the single most important favourable prognostic parameter for induction remission rate and survival duration in patients with acute myeloid leukaemia (AML). The presence of Auer bodies in 205 AML patients was studied and its relationship with five cytological and cytochemical features of leukaemic cell maturation: the presence of granules, low nuclear/cytoplasmic ratio, Sudan black positivity, esterases positivity and the maturation index was assessed in bone marrow smears of each AML patient. The results showed a significant correlation between the presence of Auer bodies and both young age and more differentiated leukaemic cells. These findings may explain their favourable prognostic significance in AML patients.

A biological hypothesis for the FAB classification of acute myeloid leukaemias.

A biological hypothesis which is based upon the response of AML blast cells to retinoic acid alone and in combinations with other differentiating agents in primary culture, is proposed for the FAB classification of Acute Myeloid Leukaemias. The present biological hypothesis accounts for the biological and clinical observations in AML.

Aspects of treatment of acute myeloid leukemia in adults.

The treatment of acute myeloid leukemia has made steady rather than spectacular progress in the past few years, principally because of improved supportive care. The use of hematopoietic growth factors may contribute to the speed of normal bone marrow recovery following treatment; however, their potential role as modifiers of drug pharmacokinetics may be equally valuable in overcoming drug resistance. The full range of expression of the phenomenon of resistance is being intensively explored, and therapeutic opportunities for overcoming the P-glycoprotein pump are now being introduced. Gene therapy and exciting immunologic manipulations are also developing rapidly, and the role of suppressor genes has reached a fascinating point in research and clinical applications.

Calciphylaxis in man.

A patient with renal failure developed widespread calcification in the thighs after the injection of iron-dextran (Imferon). This is considered as an example of calciphylaxis, a process in which calcium is laid down in parts of the body following the administration of a "challenger." Iron-dextran is a known challenger, and should be used with caution in uraemic subjects, who may be sensitized by high serum parathyroid hormone levels. Contributing factors may have been a high calcium/phosphate product, steroids, and the patient's immobility.

Triple combination of retinoic acid + low concentration of cytosine arabinoside + hexamethylene bisacetamide induces differentiation of human AML blasts in primary culture.

Differentiation induction therapy provides an alternative therapeutic approach for patients with acute myeloid leukemia (AML) who are either unsuitable for or unresponsive to conventional cytotoxic chemotherapy. The effect of a triple combination of retinoic acid (RA) + low concentration of cytosine arabinoside (Ara-C) + hexamethylene bisacetamide (HMBA) on differentiation of blasts from 24 AML patients was studied. Non-adherent mononuclear cells were seeded at a concentration of 5 x 10(5) cells per ml in 24-well tissue culture plates containing RPMI 1640 culture medium with 20 per cent fetal calf serum and 10 per cent 5637-conditioned medium and incubated with 10(-6) M retinoic acid, 10(-6) M cytosine arabinoside and/or 2 mM hexamethylene bisacetamide for six days at 37 degrees C in a humidified incubator under 5 per cent CO2. Morphological, cytochemical and functional differentiation into mature cells were induced in blasts from 22 out of the 24 AML patients following exposure to the triple combination of 10(-6) M RA + 10(-6)M Ara-C + 2 mM HMBA in primary culture. These effective results justify a clinical trial of such triple combination for AML patients who are either unsuitable for or unresponsive to conventional cytotoxic chemotherapy.

Effect of AML serum on normal human myeloid differentiation and hexamethylene bisacetamide-induced granulocytic differentiation of the human HL-60 promyelocytic leukaemic cell line.

The effect of serum from 32 AML patients on the normal human myeloid differentiation and the hexamethylene-bisacetamide induced granulocytic differentiation of HL-60 promyelocytic leukaemic cell line was studied. Nonadherent normal mononuclear marrow cells were cultured in vitro at a concentration of 5 x 10(5) cells/ml for 6 days with each of the 32 AML sera. Ten normal human AB sera were used as control. The results showed an inhibitory activity on both morphological and functional differentiation of normal human myeloid immature marrow cells by 29 out of the 32 AML sera tested. These 29 AML sera were added to cultures of HL-60 (2.5 x 10(5)/ml) leukaemia cell line which incorporated 2 mM hexamethylene-bisacetamide for 6 days. The results showed no significant inhibition of hexamethylene-bisacetamide induced granulocytic differentiation by any of the 29 AML sera. The efficacy of hexamethylene-bisacetamide in inducing differentiation in the presence of inhibitory factors suggests a possible role in the treatment of AML patients.

The effect of human recombinant granulocyte-macrophage colony stimulating factor on the proliferation and differentiation of myeloid progenitors in congenital agranulocytosis marrow cells.

Congenital agranulocytosis is a rare and frequently fatal infantile disease characterized by recurrent bacterial infections, persistent absence of neutrophils in the peripheral blood and an arrest of myeloid maturation at the promyelocyte/myelocyte stage. The effect of human recombinant Granulocyte-Macrophage colony stimulating factor (GM-CSF) alone and in combination with retinoic acid, dimethylsulphoxide or actinomycin-D on the proliferation and differentiation of bone marrow cells from a child with congenital agranulocytosis was studied. Cells were treated at a concentration of 1 x 10(5) per ml in in-vitro culture with GM-CSF alone and in combination with retinoic acid, dimethylsulphoxide or actinomycin-D for 7 days at 37 degrees C in humidified incubator containing 5% CO2 in air. GM-CSF showed a profound stimulatory effect on the proliferation of myeloid progenitors from the child bone marrow and restored colony numbers in the retinoic acid-, dimethylsulphoxide- and actinomycin-D-inhibited cultures.

Retinoic acid alone and in combination with cytosine arabinoside induces differentiation of human myelomonocytic and monoblastic leukaemic cells.

The effect of retinoic acid (RA) alone and in combination with cytosine arabinoside (Ara-C) on differentiation of fresh human myeloid leukaemic cells from patients with AML was studied. Cells from six patients: three with acute myelomonocytic leukaemia AMMoL and three with acute monoblastic leukaemia AMoL with a percentage of blasts greater than 70, were treated in an in vitro primary suspension culture with retinoic acid (10(-7) M), cytosine arabinoside (100 ng/ml) or both in combination. Non-adherent mononuclear cells were seeded at a concentration of 5 x 10(5) cells/ml in RPMI 1640 culture medium supplemented with 20 per cent fetal bovine serum and 10 per cent (PHA-LCM) phytohaemagglutinin leucocyte conditioned medium and incubated for 6 days at 37 degrees C in a humidified incubator containing 5 per cent CO2 in air. Morphological and functional differentiation into terminal mature elements was induced in all leukaemia cells of the six patients following exposure to the combination of both agents. These results suggest the potential usefulness of the combination of a differentiating agent (retinoic acid) and an antileukaemic drug (cytosine arabinoside) in the treatment of acute myeloid leukaemias: AMMoL and AMoL. This combination warrants a clinical trial.